US20120009580A1

US20120009580A1 - Methods for quantitating small rna molecules

Info

Publication number: US20120009580A1
Application number: US13/240,847
Authority: US
Inventors: Christopher K. Raymond
Original assignee: Merck Sharp and Dohme LLC
Current assignee: Merck Sharp and Dohme LLC
Priority date: 2005-01-25
Filing date: 2011-09-22
Publication date: 2012-01-12
Also published as: EP2173907A1; CA2693974A1; WO2009012433A1; US8071306B2; JP2010533501A; AU2008275930A1; EP2514839A1; US20070292878A1

Abstract

In one aspect, the present invention provides methods for amplifying a microRNA molecule to produce DNA molecules. The methods each include the steps of: (a) using primer extension to make a DNA molecule that is complementary to a target microRNA molecule; and (b) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/779,759, filed Jul. 18, 2007, which is a continuation-in-part of application Ser. No. 10/579,029, filed Nov. 19, 2008, which is the National Stage of International Application No. PCT/US2006/002591, filed Jan. 25, 2006, which claims the benefit of Provisional Application No. 60/647,178, filed Jan. 25, 2005, all of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 37790_Sequence_Final.txt. The text file is 250 KB; was created on Sep. 20, 2011; and is being submitted via EFS-Web with the filing of the specification.

FIELD OF THE INVENTION

The present invention relates to methods of amplifying and quantitating small RNA molecules.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression (Bernstein et al. (2001), Nature 409:363-6; Dykxhoorn et al. (2003) Nat. Rev. Mol. Cell. Biol. 4:457-67). The phenomenon of RNAi was first described in Caenorhabditis elegans, where injection of double-stranded RNA (dsRNA) led to efficient sequence-specific gene silencing of the mRNA that was complementary to the dsRNA (Fire et al. (1998) Nature 391:806-11). RNAi has also been described in plants as a phenomenon called post-transcriptional gene silencing (PTGS), which is likely used as a viral defense mechanism (Jorgensen (1990) Trends Biotechnol. 8:340-4; Brigneti et al. (1998) EMBO J. 17:6739-46; Hamilton & Baulcombe (1999) Science 286:950-2).
An early indication that the molecules that regulate PTGS were short RNAs processed from longer dsRNA was the identification of short 21 to 22 nucleotide dsRNA derived from the longer dsRNA in plants (Hamilton & Baulcombe (1999) Science 286:950-2). This observation was repeated in Drosophila embryo extracts where long dsRNA was found processed into 21-25 nucleotide short RNA by the RNase III type enzyme, Dicer (Elbashir et al. (2001) Nature 411:494-8; Elbashir et al. (2001) EMBO J. 20:6877-88; Elbashir et al. (2001) Genes Dev. 15:188-200). These observations led Elbashir et al. to test if synthetic 21-25 nucleotide synthetic dsRNAs function to specifically inhibit gene expression in Drosophila embryo lysates and mammalian cell culture (Elbashir et al. (2001) Nature 411:494-8; Elbashir et al. (2001) EMBO J. 20:6877-88; Elbashir et al. (2001) Genes Dev. 15:188-200). They demonstrated that small interfering RNAs (siRNAs) had the ability to specifically inhibit gene expression in mammalian cell culture without induction of the interferon response.
These observations led to the development of techniques for the reduction, or elimination, of expression of specific genes in mammalian cell culture, such as plasmid-based systems that generate hairpin siRNAs (Brummelkamp et al. (2002) Science 296:550-3; Paddison et al. (2002) Genes Dev. 16:948-58; Paddison et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:1443-8; Paul et al. 2002) Nat. Biotechnol. 20:404-8). siRNA molecules can also be introduced into cells, in vivo, to inhibit the expression of specific proteins (see, e.g., Soutschek, J., et al., Nature 432 (7014):173-178 (2004)).
siRNA molecules have promise both as therapeutic agents for inhibiting the expression of specific proteins, and as targets for drugs that affect the activity of siRNA molecules that function to regulate the expression of proteins involved in a disease state. A first step in developing such therapeutic agents is to measure the amounts of specific siRNA molecules in different cell types within an organism, and thereby construct an “atlas” of siRNA expression within the body. Additionally, it will be useful to measure changes in the amount of specific siRNA molecules in specific cell types in response to a defined stimulus, or in a disease state.
Short RNA molecules are difficult to quantitate. For example, with respect to the use of PCR to amplify and measure the small RNA molecules, most PCR primers are longer than the small RNA molecules, and so it is difficult to design a primer that has significant overlap with a small RNA molecule, and that selectively hybridizes to the small RNA molecule at the temperatures used for primer extension and PCR amplification reactions.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for amplifying a microRNA molecule to produce cDNA molecules. The methods include the steps of: (a) producing a first DNA molecule that is complementary to a target microRNA molecule using primer extension; and (b) amplifying the first DNA molecule to produce amplified DNA molecules using a universal forward primer and a reverse primer. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule. It will be understood that, in the practice of the present invention, typically numerous (e.g., millions) of individual microRNA molecules are amplified in a sample (e.g., a solution of RNA molecules isolated from living cells).
In another aspect, the present invention provides methods for measuring the amount of a target microRNA in a sample from a living organism. The methods of this aspect of the invention include the step of measuring the amount of a target microRNA molecule in a multiplicity of different cell types within a living organism, wherein the amount of the target microRNA molecule is measured by a method including the steps of: (1) producing a first DNA molecule complementary to the target microRNA molecule in the sample using primer extension; (2) amplifying the first DNA molecule to produce amplified DNA molecules using a universal forward primer and a reverse primer; and (3) measuring the amount of the amplified DNA molecules. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule.
In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO:1 to SEQ ID NO: 499, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7. The primer molecules of the invention can be used as primers for detecting mammalian microRNA target molecules, using the methods of the invention described herein.
In another aspect, the present invention provides kits for detecting at least one mammalian target microRNA, the kits comprising one or more primer sets specific for the detection of a target microRNA, each primer set comprising (1) an extension primer for producing a cDNA molecule complementary to a target microRNA, (2) a universal forward PCR primer for amplifying the cDNA molecule and (3) a reverse PCR primer for amplifying the cDNA molecule. The extension primer comprises a first portion that hybridizes to the target microRNA molecule and a second portion that includes a hybridization sequence for a universal forward PCR primer. The reverse PCR primer comprises a sequence selected to hybridize to a portion of the cDNA molecule. In some embodiments of the kit, at least one of the universal forward and reverse primers include at least one locked nucleic acid molecule. The kits of the invention may be used to practice various embodiments of the methods of the invention.
The present invention is useful, for example, for quantitating specific microRNA molecules within different types of cells in a living organism, or, for example, for measuring changes in the amount of specific microRNAs in living cells in response to a stimulus (e.g., in response to administration of a drug).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a flow chart of a representative method of the present invention;

FIG. 2 graphically illustrates the standard curves for assays specific for the detection of microRNA targets miR-95 and miR-424 as described in EXAMPLE 3;

FIG. 3A is a histogram plot showing the expression profile of miR-1 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5;

FIG. 3B is a histogram plot showing the expression profile of miR-124 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5; and

FIG. 3C is a histogram plot showing the expression profile of miR-150 across a panel of total RNA isolated from twelve tissues as described in EXAMPLE 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the foregoing, in one aspect, the present invention provides methods for amplifying a microRNA molecule to produce cDNA molecules. The methods include the steps of: (a) using primer extension to make a DNA molecule that is complementary to a target microRNA molecule; and (b) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules. In some embodiments of the method, at least one of the universal forward primer and the reverse primer comprises at least one locked nucleic acid molecule.
As used herein, the term “locked nucleic acid molecule” (abbreviated as LNA molecule) refers to a nucleic acid molecule that includes a 2′-0,4′-C-methylene-β-D-ribofuranosyl moiety. Exemplary 2′-0,4′-C-methylene-β-D-ribofuranosyl moieties, and exemplary LNAs including such moieties, are described, for example, in Petersen, M., and Wengel, J., Trends in Biotechnology 21(2):74-81 (2003) which publication is incorporated herein by reference in its entirety.
As used herein, the term “microRNA” refers to an RNA molecule that has a length in the range of from 21 nucleotides to 25 nucleotides. Some microRNA molecules (e.g., siRNA molecules) function in living cells to regulate gene expression.
Representative Method of the Invention. FIG. 1 shows a flowchart of a representative method of the present invention. In the method represented in FIG. 1, a microRNA is the template for synthesis of a complementary first DNA molecule. The synthesis of the first DNA molecule is primed by an extension primer, and so the first DNA molecule includes the extension primer and newly synthesized DNA (represented by a dotted line in FIG. 1). The synthesis of DNA is catalyzed by reverse transcriptase.
The extension primer includes a first portion (abbreviated as FP in FIG. 1) and a second portion (abbreviated as SP in FIG. 1). The first portion hybridizes to the microRNA target template, and the second portion includes a nucleic acid sequence that hybridizes with a universal forward primer, as described infra.
A quantitative polymerase chain reaction is used to make a second DNA molecule that is complementary to the first DNA molecule. The synthesis of the second DNA molecule is primed by the reverse primer that has a sequence that is selected to specifically hybridize to a portion of the target first DNA molecule. Thus, the reverse primer does not hybridize to nucleic acid molecules other than the first DNA molecule. The reverse primer may optionally include at least one LNA molecule located within the portion of the reverse primer that does not overlap with the extension primer. In FIG. 1, the LNA molecules are represented by shaded ovals.
A universal forward primer hybridizes to the 3′ end of the second DNA molecule and primes synthesis of a third DNA molecule. It will be understood that, although a single microRNA molecule, single first DNA molecule, single second DNA molecule, single third DNA molecule and single extension, forward and reverse primers are shown in FIG. 1, typically the practice of the present invention uses reaction mixtures that include numerous copies (e.g., millions of copies) of each of the foregoing nucleic acid molecules.
The steps of the methods of the present invention are now considered in more detail.
Preparation of microRNA Molecules Useful as Templates. microRNA molecules useful as templates in the methods of the invention can be isolated from any organism (e.g., eukaryote, such as a mammal) or part thereof, including organs, tissues, and/or individual cells (including cultured cells). Any suitable RNA preparation that includes microRNAs can be used, such as total cellular RNA.
RNA may be isolated from cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein. Cells of interest include wild-type cells, drug-exposed wild-type cells, modified cells, and drug-exposed modified cells.
Additional steps may be employed to remove some or all of the DNA. Cell lysis may be accomplished with a nonionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA. In one embodiment, RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (see, Chirgwin et al., 1979, Biochemistry 18:5294-5299). Separation of RNA from DNA can also be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.
If desired, RNase inhibitors may be added to the lysis buffer. Likewise, for certain cell types, it may be desirable to add a protein denaturation/digestion step to the protocol.
The sample of RNA can comprise a multiplicity of different microRNA molecules, each different microRNA molecule having a different nucleotide sequence. In a specific embodiment, the microRNA molecules in the RNA sample comprise at least 100 different nucleotide sequences. In other embodiments, the microRNA molecules of the RNA sample comprise at least 500, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 90,000, or 100,000 different nucleotide sequences.
The methods of the invention may be used to detect the presence of any microRNA. For example, the methods of the invention can be used to detect one or more of the microRNA targets described in a database such as “the miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144, which is publicly accessible on the World Wide Web at the Wellcome Trust Sanger Institute website at http://microrna.sanger.ac.uk/sequences/. A list of exemplary microRNA targets is also described in the following references: Lagos-Quintana et al., Curr. Biol. 12(9):735-9 (2002).
Synthesis of DNA Molecules Using microRNA Molecules As Templates. In the practice of the methods of the invention, first DNA molecules are synthesized that are complementary to the microRNA target molecules, and that are composed of an extension primer and newly synthesized DNA (wherein the extension primer primes the synthesis of the newly synthesized DNA). Individual first DNA molecules can be complementary to a whole microRNA target molecule, or to a portion thereof; although typically an individual first DNA molecule is complementary to a whole microRNA target molecule. Thus, in the practice of the methods of the invention, a population of first DNA molecules is synthesized that includes individual DNA molecules that are each complementary to all, or to a portion, of a target microRNA molecule.
The synthesis of the first DNA molecules is catalyzed by reverse transcriptase. Any reverse transcriptase molecule can be used to synthesize the first DNA molecules, such as those derived from Moloney murine leukemia virus (MMLV-RT), avian myeloblastosis virus (AMV-RT), bovine leukemia virus (BLV-RT), Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV-RT). A reverse transcriptase lacking RNaseH activity (e.g., SUPERSCRIPT III™ sold by Invitrogen, 1600 Faraday Avenue, P.O. Box 6482, Carlsbad, Calif. 92008) is preferred in order to minimize the amount of double-stranded cDNA synthesized at this stage. The reverse transcriptase molecule should also preferably be thermostable so that the DNA synthesis reaction can be conducted at as high a temperature as possible, while still permitting hybridization of primer to the microRNA target molecules.
Priming the Synthesis of the First DNA Molecules. The synthesis of the first DNA molecules is primed using an extension primer. Typically, the length of the extension primer is in the range of from 10 nucleotides to 100 nucleotides, such as 20 to 35 nucleotides. The nucleic acid sequence of the extension primer is incorporated into the sequence of each, synthesized, DNA molecule. The extension primer includes a first portion that hybridizes to a portion of the microRNA molecule. Typically the first portion of the extension primer includes the 3′-end of the extension primer. The first portion of the extension primer typically has a length in the range of from 6 nucleotides to 20 nucleotides, such as from 10 nucleotides to 12 nucleotides. In some embodiments, the first portion of the extension primer has a length in the range of from 3 nucleotides to 25 nucleotides.
The extension primer also includes a second portion that typically has a length of from 18 to 25 nucleotides. For example, the second portion of the extension primer can be 20 nucleotides long. The second portion of the extension primer is located 5′ to the first portion of the extension primer. The second portion of the extension primer includes at least a portion of the hybridization site for the universal forward primer. For example, the second portion of the extension primer can include all of the hybridization site for the universal forward primer, or, for example, can include as little as a single nucleotide of the hybridization site for the universal forward primer (the remaining portion of the hybridization site for the forward primer can, for example, be located in the first portion of the extension primer). An exemplary nucleic acid sequence of a second portion of an extension primer is 5′ CATGATCAGCTGGGCCAAGA 3′ (SEQ ID NO:1).
Amplification of the DNA Molecules. In the practice of the methods of the invention, the first DNA molecules are enzymatically amplified using the polymerase chain reaction. A universal forward primer and a reverse primer are used to prime the polymerase chain reaction. The reverse primer includes a nucleic acid sequence that is selected to specifically hybridize to a portion of a first DNA molecule.
The reverse primer typically has a length in the range of from 10 nucleotides to 100 nucleotides. In some embodiments, the reverse primer has a length in the range of from 12 nucleotides to 20 nucleotides. The nucleotide sequence of the reverse primer is selected to hybridize to a specific target nucleotide sequence under defined hybridization conditions. The reverse primer and extension primer are both present in the PCR reaction mixture, and so the reverse primer should be sufficiently long so that the melting temperature (Tm) is at least 50° C., but should not be so long that there is extensive overlap with the extension primer which may cause the formation of “primer dimers.” “Primer dimers” are formed when the reverse primer hybridizes to the extension primer, and uses the extension primer as a substrate for DNA synthesis, and the extension primer hybridizes to the reverse primer, and uses the reverse primer as a substrate for DNA synthesis. To avoid the formation of “primer dimers,” typically the reverse primer and the extension primer are designed so that they do not overlap with each other by more than 6 nucleotides. If it is not possible to make a reverse primer having a Tm of at least 50° C., and wherein the reverse primer and the extension primer do not overlap by more than 6 nucleotides, then it is preferable to lengthen the reverse primer (since Tm usually increases with increasing oligonucleotide length) and decrease the length of the extension primer.
The reverse primer primes the synthesis of a second DNA molecule that is complementary to the first DNA molecule. The universal forward primer hybridizes to the portion of the second DNA molecule that is complementary to the second portion of the extension primer which is incorporated into all of the first DNA molecules. The universal forward primer primes the synthesis of third DNA molecules. The universal forward primer typically has a length in the range of from 16 nucleotides to 100 nucleotides. In some embodiments, the universal forward primer has a length in the range of from 16 nucleotides to 30 nucleotides. The universal forward primer may include at least one locked nucleic acid molecule. In some embodiments, the universal forward primer includes from 1 to 25 locked nucleic acid molecules. The nucleic acid sequence of an exemplary universal forward primer is set forth in SEQ ID NO:13.
In general, the greater the number of amplification cycles during the polymerase chain reaction, the greater the amount of amplified DNA that is obtained. On the other hand, too many amplification cycles (e.g., more than 35 amplification cycles) may result in spurious and unintended amplification of non-target double-stranded DNA. Thus, in some embodiments, a desirable number of amplification cycles is between one and 45 amplification cycles, such as from one to 25 amplification cycles, or such as from five to 15 amplification cycles, or such as ten amplification cycles.
Use of LNA Molecules and Selection of Primer Hybridization Conditions. Hybridization conditions are selected that promote the specific hybridization of a primer molecule to the complementary sequence on a substrate molecule. With respect to the hybridization of a 12 nucleotide first portion of an extension primer to a microRNA, it has been found that specific hybridization occurs at a temperature of 50° C. Similarly, it has been found that hybridization of a 20 nucleotide universal forward primer to a complementary DNA molecule, and hybridization of a reverse primer (having a length in the range of from 12-20 nucleotides, such as from 14-16 nucleotides) to a complementary DNA molecule occurs at a temperature of 50° C. By way of example, it is often desirable to design extension, reverse and universal forward primers that each have a hybridization temperature in the range of from 50° C. to 60° C.
In some embodiments, LNA molecules can be incorporated into at least one of the extension primer, reverse primer, and universal forward primer to raise the Tm of one, or more, of the foregoing primers to at least 50° C. Incorporation of an LNA molecule into the portion of the reverse primer that hybridizes to the target first DNA molecule, but not to the extension primer, may be useful because this portion of the reverse primer is typically no more than 10 nucleotides in length. For example, the portion of the reverse primer that hybridizes to the target first DNA molecule, but not to the extension primer, may include at least one locked nucleic acid molecule (e.g., from 1 to 25 locked nucleic acid molecules). In some embodiments, two or three locked nucleic acid molecules are included within the first 8 nucleotides from the 5′ end of the reverse primer.
The number of LNA residues that must be incorporated into a specific primer to raise the Tm to a desired temperature mainly depends on the length of the primer and the nucleotide composition of the primer. A tool for determining the effect on Tm of one or more LNAs in a primer is available on the Internet Web site of Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark.
Although one or more LNAs can be included in any of the primers used in the practice of the present invention, it has been found that the efficiency of synthesis of cDNA is low if an LNA is incorporated into the extension primer. While not wishing to be bound by theory, LNAs may inhibit the activity of reverse transcriptase.
Detecting and Measuring the Amount of the Amplified DNA Molecules. The amplified DNA molecules can be detected and quantitated by the presence of detectable marker molecules, such as fluorescent molecules. For example, the amplified DNA molecules can be detected and quantitated by the presence of a dye (e.g., SYBR green) that preferentially or exclusively binds to double stranded DNA during the PCR amplification step of the methods of the present invention. For example, Molecular Probes, Inc. (29851 Willow Creek Road, Eugene, Oreg. 97402) sells quantitative PCR reaction mixtures that include SYBR green dye. By way of further example, another dye (referred to as “BEBO”) that can be used to label double stranded DNA produced during real-time PCR is described by Bengtsson, M., et al., Nucleic Acids Research 3/(8):e45 (Apr. 15, 2003), which publication is incorporated herein by reference. Again by way of example, a forward and/or reverse primer that includes a fluorophore and quencher can be used to prime the PCR amplification step of the methods of the present invention. The physical separation of the fluorophore and quencher that occurs after extension of the labeled primer during PCR permits the fluorophore to fluoresce, and the fluorescence can be used to measure the amount of the PCR amplification products. Examples of commercially available primers that include a fluorophore and quencher include Scorpion primers and Uniprimers, which are both sold by Molecular Probes, Inc.
Representative Uses of the Present Invention. The present invention is useful for producing cDNA molecules from microRNA target molecules. The amount of the DNA molecules can be measured which provides a measurement of the amount of target microRNA molecules in the starting material. For example, the methods of the present invention can be used to measure the amount of specific microRNA molecules (e.g., specific siRNA molecules) in living cells. Again by way of example, the present invention can be used to measure the amount of specific microRNA molecules (e.g., specific siRNA molecules) in different cell types in a living body, thereby producing an “atlas” of the distribution of specific microRNA molecules within the body. Again by way of example, the present invention can be used to measure changes in the amount of specific microRNA molecules (e.g., specific siRNA molecules) in response to a stimulus, such as in response to treatment of a population of living cells with a drug.
Thus, in another aspect, the present invention provides methods for measuring the amount of a target microRNA in a multiplicity of different cell types within a living organism (e.g., to make a microRNA “atlas” of the organism). The methods of this aspect of the invention each include the step of measuring the amount of a target microRNA molecule in a multiplicity of different cell types within a living organism, wherein the amount of the target microRNA molecule is measured by a method comprising the steps of: (1) using primer extension to make a DNA molecule complementary to the target microRNA molecule isolated from a cell type of a living organism; (2) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules, and (3) measuring the amount of the amplified DNA molecules. In some embodiments of the methods, at least one of the forward primer and the reverse primer comprises at least one locked nucleic acid molecule. The measured amounts of amplified DNA molecules can, for example, be stored in an interrogatable database in electronic form, such as on a computer-readable medium (e.g., a floppy disc).
In some embodiments, the methods may be used to discriminate between two or more mammalian target microRNA that have a similar sequence in a sample from a living organism, the method comprising the steps of: (a) producing a first DNA molecule that is complementary to the first microRNA molecule using a first extension primer specific to the first microRNA molecule; (b) amplifying the first DNA molecule to produce a first population of amplified DNA molecules using a universal forward primer and a first reverse primer; (c) producing a second DNA molecule that is complementary to the second microRNA molecule using a second extension primer specific to the second microRNA molecule; (d) amplifying the second DNA molecule to produce a second population of amplified DNA molecules using a universal forward primer and a second reverse primer; (e) measuring the amount of the first and second population of amplified DNA molecules, wherein the first and second extension primers or the first and second reverse primers differ by one or more nucleotides in the portion that is complementary to the target microRNA. This method may be used to discriminate between microRNA targets that differ by one, two, three or more nucleotides, by designing the gene-specific region of the first and second extension primers to hybridize to the region of the microRNA targets that are not identical.
In another aspect, the invention provides nucleic acid primer molecules consisting of sequence SEQ ID NO:1 to SEQ ID NO: 499, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7. The primer molecules of the invention can be used as primers for detecting mammalian microRNA target molecules, using the methods of the invention described herein.
In another aspect, the invention provides sets of nucleic acid primers consisting of SEQ ID NO:500 to SEQ ID NO: 965, as shown in TABLE 8. The sets of primer molecules of the invention can be used for the detection of microRNA target molecules from human, mouse, and rat, using the methods of the invention described herein.
In another aspect, the present invention provides kits for detecting at least one mammalian target microRNA, the kits comprising one or more primer sets specific for the detection of a target microRNA, each primer set comprising (1) an extension primer for producing a cDNA molecule complementary to a target microRNA, (2) a universal forward PCR primer, and (3) a reverse PCR primer for amplifying the cDNA molecule. The extension primer comprises a first portion that hybridizes to the target microRNA molecule and a second portion that includes a hybridization sequence for a universal forward PCR primer. The reverse PCR primer comprises a sequence selected to hybridize to a portion of the cDNA molecule. In some embodiments of the kits, at least one of the universal forward and reverse primers includes at least one locked nucleic acid molecule.
The extension primer, universal forward and reverse primers for inclusion in the kit may be designed to detect any mammalian target microRNA in accordance with the methods described herein. Nonlimiting examples of human target microRNA target molecules and exemplary target-specific extension primers and reverse primers are listed below in TABLE 1, TABLE 2, and TABLE 6. Nonlimiting examples of murine target microRNA target molecules and exemplary target-specific extension primers and reverse primers are listed below in TABLE 7. A nonlimiting example of a universal forward primer is set forth as SEQ ID NO: 13.
In certain embodiments, the kit includes a set of primers comprising an extension primer, reverse and universal forward primers for a selected target microRNA molecule that each have a hybridization temperature in the range of from 50° C. to 60° C.
In certain embodiments, the kit includes a plurality of primer sets that may be used to detect a plurality of mammalian microRNA targets, such as two microRNA targets up to several hundred microRNA targets.
In certain embodiments, the kit comprises one or more primer sets capable of detecting at least one or more of the following human microRNA target templates: of miR-1, miR-7, miR-9*, miR-10a, miR-10b, miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-22, miR-23a, miR-23b, miR-24, miR-25, miR-26a, miR-26b, miR-27a, miR-28, miR-29a, miR-29b, miR-29c, miR-30a-5p, miR-30b, miR-30c, miR-30d, miR-30e-5p, miR-30e-3p, miR-31, miR-32, miR-33, miR-34a, miR-34b, miR-34c, miR-92, miR-93, miR-95, miR-96, miR-98, miR-99a, miR-99b, miR-100, miR-101, miR-103, miR-105, miR-106a, miR-107, miR-122, miR-122a, miR-124, miR-124, miR-124a, miR-125 a, miR-125b, miR-126, miR-126*, miR-127, miR-128a, miR-128b, miR-129, miR-130a, miR-130b, miR-132, miR-133a, miR-133b, miR-134, miR-135a, miR-135b, miR-136, miR-137, miR-138, miR-139, miR-140, miR-141, miR-142-3p, miR-143, miR-144, miR-145, miR-146, miR-147, miR-148a, miR-148b, miR-149, miR-150, miR-151, miR-152, miR-153, miR-154*, miR-154, miR-155, miR-181a, miR-181b, miR-181c, miR-182*, miR-182, miR-183, miR-184, miR-185, miR-186, miR-187, miR-188, miR-189, miR-190, miR-191, miR-192, miR-193, miR-194, miR-195, miR-196a, miR-196b, miR-197, miR-198, miR-199a*, miR-199a, miR-199b, miR-200a, miR-200b, miR-200c, miR-202, miR-203, miR-204, miR-205, miR-206, miR-208, miR-210, miR-211, miR-212, miR-213, miR-213, miR-214, miR-215, miR-216, miR-217, miR-218, miR-220, miR-221, miR-222, miR-223, miR-224, miR-296, miR-299, miR-301, miR-302a*, miR-302a, miR-302b*, miR-302b, miR-302d, miR-302c*, miR-302c, miR-320, miR-323, miR-324-3p, miR-324-5p, miR-325, miR-326, miR-328, miR-330, miR-331, miR-337, miR-338, miR-339, miR-340, miR-342, miR-345, miR-346, miR-363, miR-367, miR-368, miR-370, miR-371, miR-372, miR-373*, miR-373, miR-374, miR-375, miR-376b, miR-378, miR-379, miR-380-5p, miR-380-3p, miR-381, miR-382, miR-383, miR-410, miR-412, miR-422a, miR-422b, miR-423, miR-424, miR-425, miR-429, miR-431, miR-448, miR-449, miR-450, miR-451, let7a, let7b, let7c, let7d, let7e, let7f, let7g, let7i, miR-376a, and miR-377. The sequences of the above-mentioned microRNA targets are provided in “the miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144, which is publicly accessible on the World Wide Web at the Wellcome Trust Sanger Institute website at http://microrna.sanger.ac.uk/sequences/.
Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 1, TABLE 2, and TABLE 6 below.
In another embodiment, the kit comprises one or more primer sets capable of detecting at least one or more of the following human microRNA target templates: miR-1, miR-7, miR-10b, miR-26a, miR-26b, miR-29a, miR-30e-3p, miR-95, miR-107, miR-141, miR-143, miR-154*, miR-154, miR-155, miR-181a, miR-181b, miR-181c, miR-190, miR-193, miR-194, miR-195, miR-202, miR-206, miR-208, miR-212, miR-221, miR-222, miR-224, miR-296, miR-299, miR-302c*, miR-302c, miR-320, miR-339, miR363, miR-376b, miR379, miR410, miR412, miR424, miR429, miR431, miR449, miR451, let7a, let7b, let7c, let7d, let7e, let7f, let7g, and let7i. Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 1, TABLE 2, and TABLE 6 below.
In another embodiment, the kit comprises one or more primer sets capable of detecting at least one or more of the following human, mouse or rat microRNA target templates: miR-1, miR-9, miR-18b, miR-20b, miR-92b, miR-146b, miR-181d, miR-193b, miR-194, miR-206, miR-291a-3p, miR-291b-3p, miR-301b, miR-329, miR-346, miR-351, miR-362, miR-362-3p, miR-369-5p, miR-384, miR-409-3p, miR-409-5p, miR-425-5p, miR-449b, miR-455, miR-483, miR-484, miR-485-3p, miR-485-5p, miR-486, miR-487b, miR-488, miR-489, miR-490, miR-491, miR-493-3p, miR-494, miR-495, miR-497, miR-499, miR-500, miR-501, miR-503, miR-505, miR-519a, miR-519b, miR-519c, miR-519d, miR-520a, miR-520b, miR-520d, miR-520e, miR-520f, miR-532, miR-539, miR-542-3p, miR-542-5p, miR-615, miR-652, miR-668, miR-671, miR-675-5p, miR-699, miR-721, and miR-758.
Exemplary primers for use in accordance with this embodiment of the kit are provided in TABLE 8.
In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 493, as shown in TABLE 1, TABLE 2, TABLE 6, and TABLE 7.
In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 47, 48, 49, 50, 55, 56, 81, 82, 83, 84, 91, 92, 103, 104, 123, 124, 145, 146, 193, 194, 197, 198, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 239, 240, 247, 248, 253, 254, 255, 256, 257, 258, 277, 278, 285, 286, 287, 288, 293, 294, 301, 302, 309, 310, 311, 312, 315, 316, 317, 318, 319, 320, 333, 334, 335, 336, 337, 338, 359, 360, 369, 370, 389, 390, 393, 394, 405, 406, 407, 408, 415, 416, 419, 420, 421, 422, 425, 426, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 461 and 462, as shown in TABLE 6.
In another embodiment, the kit comprises at least one oligonucleotide primer selected from the group consisting of SEQ ID NO: 500 to SEQ ID NO: 965, as shown in TABLE 8.
A kit of the invention can also provide reagents for primer extension and amplification reactions. For example, in some embodiments, the kit may further include one or more of the following components: a reverse transcriptase enzyme, a DNA polymerase enzyme, a Tris buffer, a potassium salt (e.g., potassium chloride), a magnesium salt (e.g., magnesium chloride), a reducing agent (e.g., dithiothreitol), and deoxynucleoside triphosphates (dNTPs).
In various embodiments, the kit may include a detection reagent such as SYBR green dye or BEBO dye that preferentially or exclusively binds to double stranded DNA during a PCR amplification step. In other embodiments, the kit may include a forward and/or reverse primer that includes a fluorophore and quencher to measure the amount of the PCR amplification products.
The kit optionally includes instructions for using the kit in the detection and quantitation of one or more mammalian microRNA targets. The kit can also be optionally provided in a suitable housing that is preferably useful for robotic handling in a high throughput manner.
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

Example 1

This Example describes a representative method of the invention for producing DNA molecules from microRNA target molecules.
Primer extension was conducted as follows (using InVitrogen SuperScript III® reverse transcriptase and following the guidelines that were provided with the enzyme). The following reaction mixture was prepared on ice:

- 1 μl of 10 mM dNTPs
- 1 μl of 21.1M extension primer
- 1-5 μl of target template
- 4 μl of “5×cDNA buffer”
- 1 μl of 0.1 M DTT
- 1 μl of RNAse OUT
- 1 μl of SuperScript III® enzyme
- water to 20

The mixture was incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, then cooled to room temperature and diluted 10-fold with TE (10 mM Tris, pH 7.6, 0.1 mM EDTA).
Real-time PCR was conducted using an ABI 7900 HTS detection system (Applied Biosystems, Foster City, Calif., U.S.A.) by monitoring SYBR® green fluorescence of double-stranded PCR amplicons as a function of PCR cycle number. A typical 101.11 PCR reaction mixture contained:

- 5 μl of 2×SYBR® green master mix (ABI)
- 0.8 μl of 10 μM universal forward primer
- 0.8 μl of 10 μM reverse primer
- 1.4 μl of water
- 2.0 μl of target template (10-fold diluted RT reaction)

The reaction was monitored through 40 cycles of standard “two cycle” PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products was measured.
The foregoing method was successfully used in eleven primer extension PCR assays for quantitation of endogenous microRNAs present in a sample of total RNA. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in these 11 assays are shown in TABLE 1.

TABLE 1

Target	Primer	Primer		SEQ
microRNA	number	Name	DNA sequence (5′ to 3′)	ID NO

gene-specific extension primers¹

humanb let7a	357	let7aP4	CATGATCAGCTGGGCCAAGAAACTATACAACCT	2

human miR-1	337	miR1P5	CATGATCAGCTGGGCCAAGATACATACTTCT	3

human miR-15a	344	miR15aP3	CATGATCAGCTGGGCCAAGACACAAACCATTATG	4

human miR-16	351	miR16P2	CATGATCAGCTGGGCCAAGACGCCAATATTTACGT	5

human miR-21	342	miR21P6	CATGATCAGCTGGGCCAAGATCAACATCAGT	6

human miR-24	350	miR24P5	CATGATCAGCTGGGCCAAGACTGTTCCTGCTG	7

human miR-122	222	122-E5F	CATGATCAGCTGGGCCAAGAACAAACACCATTGTCA	8

human miR-124	226	124-E5F	CATGATCAGCTGGGCCAAGATGGCATTCACCGCGTG	9

human miR-143	362	miR143P5	CATGATCAGCTGGGCCAAGATGAGCTACAGTG	10

human miR-145	305	miR145P2	CATGATCAGCTGGGCCAAGAAAGGGATTCCTGGGAA	11

human miR-155	367	miR155P3	CATGATCAGCTGGGCCAAGACCCCTATCACGAT	12

universal forward primer

	230	E5F	CATGATCAGCTGGGCCAAGA	13

RNA species-specific reverse primers²

human let7a	290	miRlet7a-	TG+AGGT+AGTAGGTTG	14
		1,2,3R

human miR-1	285	miR1-1,2R	TG+GAA+TG+TAAAGAAGTA	15

human miR-15a	287	miR15aR	TAG+CAG+CACATAATG	16

human miR-16	289	miR16-1,2R	T+AGC+AGCACGTAAA	17

human miR-21	286	miR21R	T+AG+CT+TATCAGACTGAT	18

human miR-24	288	miR24-1,2R	TGG+CTCAGTTCAGC	19

human miR-122	234	122LNAR	T+G+GAG+TGTGACAA	20

human miR-124	235	124LNAR	T+TAA+GGCACGCG	21

human miR-143	291	miR143R	TG+AGA+TGAAGCACTG	22

human miR-145	314	miR145R2	GT+CCAGTTTTCCCA	23

human miR-155	293	miR155R	T+TAA+TG+CTAATCGTGA	24

¹Universal forward primer binding sites are shown in italics. The overlap with the RNA-specific reverse primers are underlined.
²LNA molecules are preceded by a “+”. Region of overlap of the reverse primers with the corresponding extension primers are underlined.

The assay was capable of detecting microRNA in a concentration range of from 2 nM to 20 fM. The assays were linear at least up to a concentration of 2 nM of synthetic microRNA (>1,000,000 copies/cell).

Example 2

This Example describes the evaluation of the minimum sequence requirements for efficient primer-extension mediated cDNA synthesis using a series of extension primers for microRNA assays having gene specific regions that range in length from 12 to 3 base pairs.
Primer Extension Reactions. Primer extension was conducted using the target molecules miR-195 and miR-215 as follows. The target templates miR-195 and miR-215 were diluted to 1 nM RNA (100,000 copies/cell) in TE zero plus 100 ng/μl total yeast RNA. A no template control (NTC) was prepared with TE zero plus 100 ng/μl total yeast RNA.
The reverse transcriptase reactions were carried out as follows (using InVitrogen SuperScript III® reverse transcriptase and following the guidelines that were provided with the enzyme) using a series of extension primers for miR-195 (SEQ ID NO: 25-34) and a series of extension primers for miR-215 (SEQ ID NO: 35-44) the sequences of which are shown below in TABLE 2.
The following reaction mixtures were prepared on ice:

- Set 1: No Template Control
- 37.5 μl water
- 12.5 μl of 10 mM dNTPs
- 12.5 μl 0.1 mM DTT
- 50 μl of “5×cDNA buffer”
- 12.5 μl RNAse OUT
- 12.5 μl Superscript III® reverse transcriptase enzyme
- 12.5 μl 1 μg/μl Hela cell total RNA (Ambion)
- plus 50 μl of 2 μM extension primer
- plus 50 μl TEzero+yeast RNA
- Set 2: Spike-in Template
- 37.5 μl water
- 12.5 μl of 10 mM dNTPs
- 12.5 μl 0.1 mM DTT
- 50 μl of “5×cDNA buffer”
- 12.5 μl RNAse OUT
- 12.5 μl Superscript III® reverse transcriptase enzyme (InVitrogen)
- 12.5 μl 1 μg/μl Hela cell total RNA (Ambion)
- plus 50 μl of 2 μM extension primer
- plus 50 μl 1 nM RNA target template (miR-195 or miR-215)
- serially diluted in 10-fold increments

The reactions were incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, and cooled to 4° C. and diluted 10-fold with TE (10 mM Tris, pH 7.6, 0.1 mM EDTA).
Quantitative Real-Time PCR Reactions. Following reverse transcription, quadruplicate measurements of cDNA were made by quantitative real-time (qPCR) using an ABI 7900 HTS detection system (Applied Biosystems, Foster City, Calif., U.S.A.) by monitoring SYBR® green fluorescence of double-stranded PCR amplicons as a function of PCR cycle number. The following reaction mixture was prepared:

- 5 μl of 2×SYBR green master mix (ABI)
- 0.8 μl of 10 μM universal forward primer (SEQ ID NO: 13)
- 0.8 μl of 10 μM reverse primer (miR-195RP:SEQ ID NO: 45 or
- miR215RP: SEQ ID NO: 46)
- 1.4 μl of water
- 2.0 μl of target template (10-fold diluted miR-195 or miR-215 RT reaction)

Quantitative real-time PCR was performed for each sample in quadruplicate, using the manufacturer's recommended conditions. The reactions were monitored through 40 cycles of standard “two cycle” PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products were measured and disassociation curves were generated. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in the miR-195 and miR-215 assays are shown below in TABLE 2. The assay results for miR-195 are shown below in TABLE 3 and the assay results for miR-215 are shown below in TABLE 4.

TABLE 2

Target	Primer			SEQ ID
microRNA	number	Primer Name	DNA sequence (5′ to 3′)	NO:

gene-specific extension primers¹

miR-195	646	mir195-GS1	CATGATCAGCTGGGCCAAGAGCCAATATTTCT	25

miR-195	647	mir195-GS2	CATGATCAGCTGGGCCAAGAGCCAATATTTC	26

miR-195	648	mir195-GS3	CATGATCAGCTGGGCCAAGAGCCAATATTT	27

miR-195	649	mir195-GS4	CATGATCAGCTGGGCCAAGAGCCAATATT	28

miR-195	650	mir195-GS5	CATGATCAGCTGGGCCAAGAGCCAATAT	29

miR-195	651	mir195-GS6	CATGATCAGCTGGGCCAAGAGCCAATA	30

miR-195	652	mir195-GS7	CATGATCAGCTGGGCCAAGAGCCAAT	31

miR-195	653	mir195-GS8	CATGATCAGCTGGGCCAAGAGCCAA	32

miR-195	654	mir195-GS9	CATGATCAGCTGGGCCAAGAGCCA	33

miR-195	655	mir195-GS10	CATGATCAGCTGGGCCAAGAGCC	34

miR-215	656	mir215-GS1	CATGATCAGCTGGGCCAAGAGTCTGTCAATTC	35

miR-215	657	mir215-GS2	CATGATCAGCTGGGCCAAGAGTCTGTCAATT	36

miR-215	658	mir215-GS3	CATGATCAGCTGGGCCAAGAGTCTGTCAAT	37

miR-215	659	mir215-GS4	CATGATCAGCTGGGCCAAGAGTCTGTCAA	38

miR-215	660	mir215-GS5	CATGATCAGCTGGGCCAAGAGTCTGTCA	39

miR-215	661	mir215-GS6	CATGATCAGCTGGGCCAAGAGTCTGTC	40

miR-215	662	mir215-GS7	CATGATCAGCTGGGCCAAGAGTCTGT	41

miR-215	663	mir215-GS8	CATGATCAGCTGGGCCAAGAGTCTG	42

miR-215	664	mir215-GS9	CATGATCAGCTGGGCCAAGAGTCT	43

miR-215	665	mir215-GS10	CATGATCAGCTGGGCCAAGAGTC	44

RNA species-specific reverse primers²

miR-195	442	mir195RP	T+AGC+AGCACAGAAAT	45

miR-215	446	mir215RP	A+T+GA+CCTATGAATTG	46

¹Universal forward primer binding sites are shown in italics.
²The “+” symbol precedes the LNA molecules.

Results:
The sensitivity of each assay was measured by the cycle threshold (Ct) value which is defined as the cycle count at which fluorescence was detected in an assay containing microRNA target template. The lower this Ct value (e.g. the fewer number of cycles), the more sensitive was the assay. For microRNA samples, it was generally observed that while samples that contain template and no template controls both eventually cross the detection threshold, the samples with template do so at a much lower cycle number. The ΔCt value is the difference between the number of cycles (Ct) between template containing samples and no template controls, and serves as a measure of the dynamic range of the assay. Assays with a high dynamic range allow measurements of very low microRNA copy numbers. Accordingly, desirable characteristics of a microRNA detection assay include high sensitivity (low Ct value) and broad dynamic range (ΔCt≧12) between the signal of a sample containing target template and a no template background control sample.
The results of the miR195 and miR215 assays using extension primers having a gene specific portion ranging in size from 12 nucleotides to 3 nucleotides are shown below in TABLE 3 and TABLE 4, respectively. The results of these experiments unexpectedly demonstrate that gene-specific priming sequences as short as 3 nucleotides exhibit template specific priming. For both the miR-195 assay sets (shown in TABLE 3) and the miR-215 assay sets (shown in TABLE 4), the results demonstrate that the dynamic range (ΔCt) for both sets of assays are fairly consistent for extension primers having gene specific regions that are greater or equal to 8 nucleotides in length. The dynamic range of the assay (ΔCt) begins to decrease for extension primers having gene specific regions below 8 nucleotides, with a reduction in assay specificity below 7 nucleotides in the miR-195 assays, and below 6 nucleotides in the miR-215 assays. A melting point analysis of the miR-215 samples demonstrated that even at 3 nucleotides, there is specific PCR product present in the plus template samples (data not shown). Taken together, these data demonstrate that the gene specific region of extension primers is ideally ≧8 nucleotides, but can be as short as 3 nucleotides in length.

TABLE 3

MIR195 ASSAY RESULTS

GS Primer	Ct: No	Ct: Plus
Length	Template Control	Template	Δ Ct

12	34.83	20.00	14.82
12	34.19	19.9	14.3
11	40.0	19.8	20.2
10	36.45	21.2	15.2
9	36.40	22.2	14.2
8	40.0	23.73	16.27
7	36.70	25.96	10.73
6	30.95	26.58	4.37
5	30.98	31.71	−0.732
4	32.92	33.28	−0.364
3	35.98	35.38	−0.605

Ct = the cycle count where the fluorescence exceeds the threshold of detection.
Δ Ct = the difference between the Ct value with template and no template.

TABLE 4

MIR215 ASSAY RESULTS

GS Primer	Ct: No	Ct: Plus
Length	Template Control	Template	Δ Ct

12	33.4	13.57	19.83
12	33.93	14.15	19.77
11	35.51	15.76	19.75
10	35.33	15.49	19.84
9	36.02	16.84	19.18
8	35.79	17.07	18.72
7	32.29	17.58	14.71
6	34.38	20.62	13.75
5	34.41	28.65	5.75
4	36.36	33.92	2.44
3	35.09	33.38	1.70

Ct = the cycle count where the fluorescence exceeds the threshold of detection.
Δ Ct = the difference between the Ct value with template and no template.

Example 3

This Example describes assays and primer sets designed for quantitative analysis of human microRNA expression patterns.
Primer Design:
microRNA target templates: the sequence of the target templates as described herein are publicly available accessible on the World Wide Web at the Wellcome Trust Sanger Institute Web site in the “miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111, and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144.
Extension primers: gene specific primers for primer extension of a microRNA to form a cDNA followed by quantitative PCR (qPCR) amplification were designed to (1) convert the RNA template into cDNA; (2) to introduce a “universal” PCR binding site (SEQ ID NO:1) to one end of the cDNA molecule; and (3) to extend the length of the cDNA to facilitate subsequent monitoring by qPCR.
Reverse primers: unmodified reverse primers and locked nucleic acid (LNA) containing reverse primers (RP) were designed to quantify the primer-extended, full length cDNA in combination with a generic universal forward primer (SEQ ID NO:13). For the locked nucleic acid containing reverse primers, two or three LNA modified bases were substituted within the first 8 nucleotides from the 5′ end of the reverse primer oligonucleotide, as shown below in the exemplary reverse primer sequences provided in TABLE 6. The LNA base substitutions were selected to raise the predicted Tm of the primer by the highest amount, and the final predicted Tm of the selected primers were specified to be preferably less than or equal to 55° C.
An example describing an assay utilizing an exemplary set of primers the detection of miR-95 and miR-424 is described below.
Primer Extension Reactions: primer extension was conducted using DNA templates corresponding to miR-95 and miR-424 as follows. The DNA templates were diluted to 0 nM, 1 nM, 100 pM, 10 pM, and 1 pM dilutions in TE zero (10 mM Tris pH 7.6, 0.1 mM EDTA) plus 100 ng/μl yeast total RNA (Ambion, Austin, Tex.).
The reverse transcriptase reactions were carried out using the following primers:
Extension primers: (diluted to 500 nM)

miR-95GSP
CATGATCAGCTGGGCCAAGATGCTCAATAA	(SEQ ID NO: 123)

miR-424GSP
CATGATCAGCTGGGCCAAGATTCAAAACAT	(SEQ ID NO: 415)

Reverse primers: (diluted to 10 mM)

	miR-95_RP4
	TT+CAAC+GGGTATTTATTGA	(SEQ ID NO: 124)

	miR-424RP2
	C+AG+CAGCAATTCATGTTTT	(SEQ ID NO: 416)

Reverse Transcription (per reaction):
2 μl water
2 μl of “5×cDNA buffer” (InVitrogen, Carlsbad, Calif.)
0.5 μl of 0.1 mM DTT (InVitrogen, Carlsbad, Calif.)
0.5 μl of 10 mM dNTPs (InVitrogen, Carlsbad, Calif.)
0.5 μl RNAse OUT (InVitrogen, Carlsbad, Calif.)
0.5 μl Superscript III® reverse transcriptase enzyme (InVitrogen, Carlsbad, Calif.)
2 μl of extension primer plus 2 μl of template dilution
The reactions were mixed and incubated at 50° C. for 30 minutes, then 85° C. for 5 minutes, and cooled to 4° C. and diluted 10-fold with TE zero.
Quantitative Real-Time PCR Reactions (per reaction):

- 5 μl 12×SYBR mix (Applied Biosystems, Foster City, Calif.)
- 1.4 μl water
- 0.8 μl universal primer (CATGATCAGCTGGGCCAAGA (SEQ ID NO: 13))
- 2.0 μl of diluted reverse transcription (RT) product from above.

Quantitative real-time PCR was performed for each sample in quadruplicate, using the manufacturer's recommended conditions. The reactions were monitored through 40 cycles of standard “two cycle” PCR (95° C.-15 sec, 60° C.-60 sec) and the fluorescence of the PCR products were measured and disassociation curves were generated. The DNA sequences of the extension primers, the universal forward primer sequence, and the LNA substituted reverse primers, used in the representative miR-95 and miR-424 assays as well as primer sets for 212 different human microRNA templates are shown below in TABLE 6. Primer sets for assays requiring extensive testing and design modification to achieve a sensitive assay with a high dynamic range are indicated in TABLE 6 with the symbol # following the primer name.
Results:
TABLE 5 shows the Ct values (averaged from four samples) from the miR-95 and miR-424 assays, which are plotted in the graph shown in FIG. 2. The results of these assays are provided as representative examples in order to explain the significance of the assay parameters shown in TABLE 6 designated as slope (column 6), intercept (column 7) and background (column 8).
As shown in TABLE 5, the Ct value for each template at various concentrations is provided. The Ct values (x-axis) are plotted as a function of template concentration (y-axis) to generate a standard curve for each assay, as shown in FIG. 2. The slope and intercept define the assay measurement characteristics that permit an estimation of number of copies/cell for each microRNA. For example, when the Ct values for 50 μg total RNA input for the miR-95 assay are plotted, a standard curve is generated with a slope and intercept of −0.03569 and 9.655, respectively. When these standard curve parameters are applied to the Ct of an unknown sample (x), they yield log 10 (copies/20 pg total RNA) (y). Because the average cell yields 20 pg of total RNA, these measurements equate to copies of microRNA/cell. The background provides an estimate of the minimum copy number that can be measured in a sample and is computed by inserting the no template control (NTC) value into this equation. In this example, as shown in TABLE 6, miR-95 yields a background of 1.68 copies/20 pg at 50 μg of RNA input.
As further shown in TABLE 6, reverse primers that do not contain LNA may also be used in accordance with the methods of the invention. See, e.g., SEQ ID NO:494-499. The sensitivity and dynamic range of the assays using non-LNA containing reverse primers SEQ ID NO:494-499, yielded similar results to the corresponding assays using LNA-containing reverse primers.

TABLE 5

Ct Values (averaged from four samples)

Template concentration

	10 nM	1 nM	0.1 nM	0.01 nM	0.001 nM	NTC

copies/20 pg	500,000	50,000	5000	500	50
RNA (50 μg input)
copies/20 pg	5,000,000	500,000	50,000	5000	500
RNA (5 μg input)
miR-95	11.71572163	14.17978	17.46353	19.97259	23.33171	27.44383
miR-424	10.47708975	12.76806	15.69251	18.53729	21.56897	23.2813
log10 (copies	5.698970004	4.69897	3.69897	2.69897	1.69897
for 50 μg input)

TABLE 6

PRIMERS TO DETECT HUMAN MICRORNA TARGET TEMPLATES

Human
Target		Background
micro		RNA input

RNA	Extension Primer Name	Extension Primer Sequence	Reverse Primer Name	Reverse Primer Sequence	Slope	Intercept	50 ug	5 ug

miR-1	miR1GSP10^#	CATGATCAGCTGGGCCAAGATACATACTTC	miR-1RP^#	T+G+GAA+TG+TAAAGAAGT	−0.2758	8.3225	2.44	24.36
		SEQ ID NO: 47		SEQ ID NO: 48

miR-7	miR-7GSP10^#	CATGATCAGCTGGGCCAAGACAACAAAATC	miR-7_RP6^#	T+GGAA+GACTAGTGATTTT	−0.2982	10.435	11.70	116.99
		SEQ ID NO: 49		SEQ ID NO: 50

miR-9*	miR-9*GSP	CATGATCAGCTGGGCCAAGAACTTTCGGTT	miR-9*RP	TAAA+GCT+AGATAACCG	−0.2405	8.9145	3.71	37.15
		SEQ ID NO: 51		SEQ ID NO: 52

miR-10a	miR-10aGSP	CATGATCAGCTGGGCCAAGACACAAATTCG	miR-10aRP	T+AC+CCTGTAGATCCG	−0.2755	8.6976	0.09	0.94
		SEQ ID NO: 53		SEQ ID NO: 54

miR-10b	miR-	CATGATCAGCTGGGCCAAGAACAAATTCGGT	miR-	TA+CCC+TGT+AGAACCGA	−0.3505	8.7109	0.55	5.52
	10b_GSP11^#	SEQ ID NO: 55	10b_RP2^#	SEQ ID NO: 56

miR-15a	miR-15aGSP	CATGATCAGCTGGGCCAAGACACAAACCAT	miR-15aRP	T+AG+CAGCACATAATG	−0.2831	8.4519	4.40	44.01
		SEQ ID NO: 57		SEQ ID NO: 58

miR-15b	miR-15bGSP2	CATGATCAGCTGGGCCAAGATGTAAACCA	miR-15bRP	T+AG+CAGCACATCAT	−0.2903	8.4206	0.18	1.84
		SEQ ID NO: 59		SEQ ID NO: 60

miR-16	miR-16GSP2	CATGATCAGCTGGGCCAAGACGCCAATAT	miR-16RP	T+AG+CAGCACGTAAA	−0.2542	9.3689	1.64	16.42
		SEQ ID NO: 61		SEQ ID NO: 62

miR-17-	miR-17-3pGSP	CATGATCAGCTGGGCCAAGAACAAGTGCCT	miR-17-3pRP	A+CT+GCAGTGAAGGC	−0.2972	8.2625	1.08	10.78
3p		SEQ ID NO: 63		SEQ ID NO: 64

miR-17-	miR-17-	CATGATCAGCTGGGCCAAGAACTACCTGC	miR-17-5pRP	C+AA+AGTGCTTACAGTG	−0.2956	7.9101	0.13	1.32
5p	5pGSP2	SEQ ID NO: 65		SEQ ID NO: 66

miR-19a	miR-19aGSP2	CATGATCAGCTGGGCCAAGATCAGTTTTG	miR-19aRP	TG+TG+CAAATCTATGC	−0.2984	9.461	0.02	0.23
		SEQ ID NO: 67		SEQ ID NO: 68

miR-19b	miR-19bGSP	CATGATCAGCTGGGCCAAGATCAGTTTTGC	miR-19bRP	TG+TG+CAAATCCATG	−0.294	8.1434	2.26	22.55
		SEQ ID NO: 69		SEQ ID NO: 70

miR-20	miR-20GSP3	CATGATCAGCTGGGCCAAGACTACCTGC	miR-20RP	T+AA+AGTGCTTATAGTGCA	−0.2979	7.9929	0.16	1.60
		SEQ ID NO: 71		SEQ ID NO: 72

miR-21	miR-21GSP2	CATGATCAGCTGGGCCAAGATCAACATCA	miR-21RP	T+AG+CTTATCAGACTGATG	−0.2849	8.1624	1.80	17.99
		SEQ ID NO: 73		SEQ ID NO: 74

miR-23a	miR-23aGSP	CATGATCAGCTGGGCCAAGAGGAAATCCCT	miR-23aRP	A+TC+ACATTGCCAGG	−0.3172	9.4253	2.41	24.08
		SEQ ID NO: 75		SEQ ID NO: 76

miR-23b	miR-23bGSP	CATGATCAGCTGGGCCAAGAGGTAATCCCT	miR-23bRP	A+TC+ACATTGCCAGG	−0.2944	9.0985	5.39	53.85
		SEQ ID NO: 77		SEQ ID NO: 78

miR-25	miR-25GSP	CATGATCAGCTGGGCCAAGATCAGACCGAG	miR-25RP	C+AT+TGCACTTGTCTC	−0.3009	8.2482	1.52	15.19
		SEQ ID NO: 79		SEQ ID NO: 80

miR-26a	miR-26aGSP9^#	CATGATCAGCTGGGCCAAGAGCCTATCCT	miR-	TT+CA+AGTAATCCAGGAT	−0.2807	8.558	0.26	2.56
		SEQ ID NO: 81	26aRP2^#	SEQ ID NO: 82

miR-26b	miR-26bGSP9^#	CATGATCAGCTGGGCCAAGAAACCTATCC	miR-	TT+CA+AGT+AATTCAGGAT	−0.2831	8.7885	0.37	3.67
		SEQ ID NO: 83	26bRP2^#	SEQ ID NO: 84

miR-27a	miR-27aGSP	CATGATCAGCTGGGCCAAGAGCGGAACTTA	miR-27aRP	TT+CA+CAGTGGCTAA	−0.2765	9.5239	5.15	51.51
		SEQ ID NO: 85		SEQ ID NO: 86

miR-27b	miR-27bGSP	CATGATCAGCTGGGCCAAGAGCAGAACTTA	miR-27bRP	TT+CA+CAGTGGCTAA	−0.28	9.5483	5.97	59.71
		SEQ ID NO: 87		SEQ ID NO: 88

miR-28	miR-28GSP	CATGATCAGCTGGGCCAAGACTCAATAGAC	miR-28RP	A+AG+GAGCTCACAGT	−0.3226	10.071	7.19	71.87
		SEQ ID NO: 89		SEQ ID NO: 90

miR-29a	miR-29aGSP8^#	CATGATCAGCTGGGCCAAGAAACCGATT	miR-	T+AG+CACCATCTGAAAT	−0.29	8.8731	0.04	0.38
		SEQ ID NO: 91	29aRP2^#	SEQ ID NO: 92

miR-29b	miR-29bGSP2	CATGATCAGCTGGGCCAAGAAACACTGAT	miR-29bRP2	T+AG+CACCATTTGAAATCAG	−0.3162	9.6276	3.56	35.57
		SEQ ID NO: 93		SEQ ID NO: 94

miR-30a-	miR-30a-	CATGATCAGCTGGGCCAAGACTTCCAGTCG	miR-30a-	T+GT+AAACATCCTCGAC	−0.2772	9.0694	1.92	19.16
5p	5pGSP	SEQ ID NO: 95	5pRP	SEQ ID NO: 96

miR-30b	miR-30bGSP	CATGATCAGCTGGGCCAAGAAGCTGAGTGT	miR-30bRP	TGT+AAA+CATCCTACACT	−0.2621	8.5974	0.11	1.13
		SEQ ID NO: 97		SEQ ID NO: 98

miR-30c	miR-30cGSP	CATGATCAGCTGGGCCAAGAGCTGAGAGTG	miR-30cRP	TGT+AAA+CATCCTACACT	−0.2703	8.699	0.15	1.48
		SEQ ID NO: 99		SEQ ID NO: 100

miR-30d	miR-30dGSP	CATGATCAGCTGGGCCAAGACTTCCAGTCG	miR-30dRP	T+GTAAA+CATCCCCG	−0.2506	9.3875	0.23	2.31
		SEQ ID NO: 101		SEQ ID NO: 102

miR-30e-	miR-30e-	CATGATCAGCTGGGCCAAGAGCTGTAAAC	miR-30e-	CTTT+CAGT+CGGATGTTT	−0.325	11.144	6.37	63.70
3p	3pGSP9^#	SEQ ID NO: 103	3pRP5^#	SEQ ID NO: 104

miR-30e-	miR-30e-	CATGATCAGCTGGGCCAAGATCCAGTCAAG	miR-30e-	TG+TAAA+CATCCTTGAC	−0.2732	8.1604	8.50	85.03
5p	5pGSP	SEQ ID NO: 105	5pRP	SEQ ID NO: 106

miR-31	miR-31GSP	CATGATCAGCTGGGCCAAGACAGCTATGCC	miR-31RP	G+GC+AAGATGCTGGC	−0.3068	8.2605	3.74	37.43
		SEQ ID NO: 107		SEQ ID NO: 108

miR-32	miR-32GSP	CATGATCAGCTGGGCCAAGAGCAACTTAGT	miR-32RP	TATTG+CA+CATTACTAAG	−0.2785	8.9581	0.39	3.93
		SEQ ID NO: 109		SEQ ID NO: 110

miR-33	miR-33GSP2	CATGATCAGCTGGGCCAAGACAATGCAAC	miR-33RP	G+TG+CATTGTAGTTGC	−0.3031	8.42	2.81	28.14
		SEQ ID NO: 111		SEQ ID NO: 112

miR-34a	miR-34aGSP	CATGATCAGCTGGGCCAAGAAACAACCAGC	miR-34aRP	T+GG+CAGTGTCTTAG	−0.3062	9.1522	2.40	23.99
		SEQ ID NO: 113		SEQ ID NO: 114

miR-34b	miR-34bGSP	CATGATCAGCTGGGCCAAGACAATCAGCTA	miR-34bRP	TA+GG+CAGTGTCATT	−0.3208	9.054	0.04	0.37
		SEQ ID NO: 115		SEQ ID NO: 116

miR-34c	miR-34cGSP	CATGATCAGCTGGGCCAAGAGCAATCAGCT	miR-34cRP	A+GG+CAGTGTAGTTA	−0.2995	10.14	1.08	10.83
		SEQ ID NO: 117		SEQ ID NO: 118

miR-92	miR-92GSP	CATGATCAGCTGGGCCAAGACAGGCCGGGA	miR-92RP	T+AT+TGCACTTGTCCC	−0.3012	8.6908	8.92	89.17
		SEQ ID NO: 119		SEQ ID NO: 120

miR-93	miR-93GSP	CATGATCAGCTGGGCCAAGACTACCTGCAC	miR-93RP	AA+AG+TGCTGTTCGT	−0.3025	7.9933	4.63	46.30
		SEQ ID NO: 121		SEQ ID NO: 122

miR-95	miR-95GSP^#	CATGATCAGCTGGGCCAAGATGCTCAATAA	miR-	TT+CAAC+GGGTATTTATTGA	−0.3436	9.655	1.68	16.80
		SEQ ID NO: 123	95_RP4^#	SEQ ID NO: 124

miR-96	miR-96GSP	CATGATCAGCTGGGCCAAGAGCAAAAATGT	miR-96RP	T+TT+GGCACTAGCAC	−0.2968	9.2611	0.00	0.05
		SEQ ID NO: 125		SEQ ID NO: 126

miR-98	miR-98GSP	CATGATCAGCTGGGCCAAGAAACAATACAA	miR-98RP	TGA+GGT+AGTAAGTTG	−0.2797	9.5654	1.05	10.48
		SEQ ID NO: 127		SEQ ID NO: 128

miR-99a	miR-99aGSP	CATGATCAGCTGGGCCAAGACACAAGATCG	miR-99aRP	A+AC+CCGTAGATCCG	−0.2768	8.781	0.21	2.08
		SEQ ID NO: 129		SEQ ID NO: 130

miR-99b	miR-99bGSP	CATGATCAGCTGGGCCAAGACGCAAGGTCG	miR-99bRP	C+AC+CCGTAGAACCG	−0.2747	7.9855	0.25	2.53
		SEQ ID NO: 131		SEQ ID NO: 132

miR-100	miR-100GSP	CATGATCAGCTGGGCCAAGACACAAGTTCG	miR-100RP	A+AC+CCGTAGATCCG	−0.2902	8.669	0.04	0.35
		SEQ ID NO: 133		SEQ ID NO: 134

miR-101	miR-101GSP	CATGATCAGCTGGGCCAAGACTTCAGTTAT	miR-101RP	TA+CAG+TACTGTGATAACT	−0.3023	8.2976	0.46	4.63
		SEQ ID NO: 135		SEQ ID NO: 136

miR-103	miR-103GSP	CATGATCAGCTGGGCCAAGATCATAGCCCT	miR-103RP	A+GC+AGCATTGTACA	−0.3107	8.5776	0.02	0.21
		SEQ ID NO: 137		SEQ ID NO: 138

miR-105	miR-105GSP	CATGATCAGCTGGGCCAAGAACAGGAGTCT	miR-105RP	T+CAAA+TGCTCAGACT	−0.2667	8.9832	0.93	9.28
		SEQ ID NO: 139		SEQ ID NO: 140

miR-106a	miR-106aGSP	CATGATCAGCTGGGCCAAGAGCTACCTGCA	miR-106aRP	AAA+AG+TGCTTACAGTG	−0.3107	8.358	0.03	0.31
		SEQ ID NO: 141		SEQ ID NO: 142

miR-106b	miR-106bGSP	CATGATCAGCTGGGCCAAGAATCTGCACTG	miR-106bRP	T+AAAG+TGCTGACAGT	−0.2978	8.7838	0.10	1.04
		SEQ ID NO: 143		SEQ ID NO: 144

miR-107	miR-107GSP8^#	CATGATCAGCTGGGCCAAGATGATAGCC	miR-	A+GC+AGCATTGTACAG	−0.304	9.1666	0.34	3.41
		SEQ ID NO: 145	107RP2^#	SEQ ID NO: 146

miR-122a	miR-122aGSP	CATGATCAGCTGGGCCAAGAACAAACACCA	miR-122aRP	T+GG+AGTGTGACAAT	−0.3016	8.1479	0.06	0.58
		SEQ ID NO: 147		SEQ ID NO: 148

miR-124a	miR-124aGSP	CATGATCAGCTGGGCCAAGATGGCATTCAC	miR-124aRP	T+TA+AGGCACGCGGT	−0.3013	8.6906	0.56	5.63
		SEQ ID NO: 149		SEQ ID NO: 150

miR-125a	miR-125aGSP	CATGATCAGCTGGGCCAAGACACAGGTTAA	miR-125aRP	T+CC+CTGAGACCCTT	−0.2938	8.6754	0.09	0.91
		SEQ ID NO: 151		SEQ ID NO: 152

miR-125b	miR-125bGSP	CATGATCAGCTGGGCCAAGATCACAAGTTA	miR-125bRP	T+CC+CTGAGACCCTA	−0.283	8.1251	0.20	1.99
		SEQ ID NO: 153		SEQ ID NO: 154

miR-126	miR-126GSP	CATGATCAGCTGGGCCAAGAGCATTATTAC	miR-126RP	T+CG+TACCGTGAGTA	−0.26	8.937	0.18	1.80
		SEQ ID NO: 155		SEQ ID NO: 156

miR-126*	miR-126*GSP3	CATGATCAGCTGGGCCAAGACGCGTACC	miR-126*RP	C+ATT+ATTA+CTTTTGGTACG	−0.2969	8.184	3.58	35.78
		SEQ ID NO: 157		SEQ ID NO: 158

miR-127	miR-127GSP	CATGATCAGCTGGGCCAAGAAGCCAAGCTC	miR-127RP	T+CG+GATCCGTCTGA	−0.2432	9.1013	1.11	11.13
		SEQ ID NO: 159		SEQ ID NO: 160

miR-128a	miR-128aGSP	CATGATCAGCTGGGCCAAGAAAAAGAGACC	miR-128aRP	T+CA+CAGTGAACCGG	−0.2866	8.0867	0.16	1.60
		SEQ ID NO: 161		SEQ ID NO: 162

miR-128b	miR-128bGSP	CATGATCAGCTGGGCCAAGAGAAAGAGACC	miR-128bRP	T+CA+CAGTGAACCGG	−0.2923	8.0608	0.07	0.74
		SEQ ID NO: 163		SEQ ID NO: 164

miR-129	miR-129GSP	CATGATCAGCTGGGCCAAGAGCAAGCCCAG	miR-129RP	CTTTT+TG+CGGTCTG	−0.2942	9.7731	0.88	8.85
		SEQ ID NO: 165		SEQ ID NO: 166

miR-130a	miR-130aGSP	CATGATCAGCTGGGCCAAGAATGCCCTTTT	miR-130aRP	C+AG+TGCAATGTTAAAAG	−0.2943	8.7465	1.28	12.78
		SEQ ID NO: 167		SEQ ID NO: 168

miR-130b	miR-130bGSP	CATGATCAGCTGGGCCAAGAATGCCCTTTC	miR-130bRP	C+AG+TGCAATGATGA	−0.2377	9.1403	3.14	31.44
		SEQ ID NO: 169		SEQ ID NO: 170

miR-132	miR-132GSP	CATGATCAGCTGGGCCAAGACGACCATGGC	miR-132RP	T+AA+CAGTCTACAGCC	−0.2948	8.1167	0.11	1.13
		SEQ ID NO: 171		SEQ ID NO: 172

miR-133a	miR-133aGSP	CATGATCAGCTGGGCCAAGAACAGCTGGTT	miR-133aRP	T+TG+GTCCCCTTCAA	−0.295	9.3679	0.10	1.04
		SEQ ID NO: 173		SEQ ID NO: 174

miR-133b	miR-133bGSP	CATGATCAGCTGGGCCAAGATAGCTGGTTG	miR-133bRP	T+TG+GTCCCCTTCAA	−0.3062	8.3649	0.02	0.18
		SEQ ID NO: 175		SEQ ID NO: 176

miR-134	miR-134GSP	CATGATCAGCTGGGCCAAGACCCTCTGGTC	miR-134RP	T+GT+GACTGGTTGAC	−0.2965	9.0483	0.14	1.39
		SEQ ID NO: 177		SEQ ID NO: 178

miR-135a	miR-135aGSP	CATGATCAGCTGGGCCAAGATCACATAGGA	miR-135aRP	T+AT+GGCTTTTTATTCCT	−0.2914	8.092	1.75	17.50
		SEQ ID NO: 179		SEQ ID NO: 180

miR-135b	miR-135bGSP	CATGATCAGCTGGGCCAAGACACATAGGAA	miR-135bRP	T+AT+GGCTTTTCATTCC	−0.2962	7.8986	0.05	0.49
		SEQ ID NO: 181		SEQ ID NO: 182

miR-136	miR-136GSP	CATGATCAGCTGGGCCAAGATCCATCATCA	miR-136RP	A+CT+CCATTTGTTTTGATG	−0.3616	10.229	0.68	6.77
		SEQ ID NO: 183		SEQ ID NO: 184

miR-137	miR-137GSP	CATGATCAGCTGGGCCAAGACTACGCGTAT	miR-137RP	T+AT+TGCTTAAGAATACGC	−0.2876	8.234	8.57	85.71
		SEQ ID NO: 185		SEQ ID NO: 186

miR-138	miR-138GSP2	CATGATCAGCTGGGCCAAGACGGCCTGAT	miR-138RP	A+GC+TGGTGTTGTGA	−0.3023	9.0814	0.22	2.19
		SEQ ID NO: 187		SEQ ID NO: 188

miR-139	miR-139GSP	CATGATCAGCTGGGCCAAGAAGACACGTGC	miR-139RP	T+CT+ACAGTGCACGT	−0.2983	8.1141	6.92	69.21
		SEQ ID NO: 189		SEQ ID NO: 190

miR-140	miR-140GSP	CATGATCAGCTGGGCCAAGACTACCATAGG	miR-140RP	A+GT+GGTTTTACCCT	−0.2312	8.3231	0.13	1.34
		SEQ ID NO: 191		SEQ ID NO: 192

miR-141	miR-141GSP9^#	CATGATCAGCTGGGCCAAGACCATCTTTA	miR-	TAA+CAC+TGTCTGGTAA	−0.2805	9.6671	0.13	1.26
		SEQ ID NO: 193	141RP2^#	SEQ ID NO: 194

miR-142-	miR-142-	CATGATCAGCTGGGCCAAGATCCATAAA	miR-142-	TGT+AG+TGTTTCCTACT	−0.2976	8.4046	0.03	0.27
3p	3pGSP3	SEQ ID NO: 195	3pRP	SEQ ID NO: 196

miR-143	miR-143GSP8^#	CATGATCAGCTGGGCCAAGATGAGCTAC	miR-	T+GA+GATGAAGCACTG	−0.3008	9.2675	0.37	3.71
		SEQ ID NO: 197	143RP2^#	SEQ ID NO: 198

miR-144	miR-144GSP2	CATGATCAGCTGGGCCAAGACTAGTACAT	miR-144RP	TA+CA+GTAT+AGATGATG	−0.2407	9.4441	0.95	9.52
		SEQ ID NO: 199		SEQ ID NO: 200

miR-145	miR-145GSP2	CATGATCAGCTGGGCCAAGAAAGGGATTC	miR-145RP	G+TC+CAGTTTTCCCA	−0.2937	8.0791	0.39	3.86
		SEQ ID NO: 201		SEQ ID NO: 202

miR-146	miR-146GSP3	CATGATCAGCTGGGCCAAGAAACCCATG	miR-146RP	T+GA+GAACTGAATTCCA	−0.2861	8.8246	0.08	0.75
		SEQ ID NO: 203		SEQ ID NO: 204

miR-147	miR-147GSP	CATGATCAGCTGGGCCAAGAGCAGAAGCAT	miR-147RP	G+TG+TGTGGAAATGC	−0.2989	8.8866	1.65	16.47
		SEQ ID NO: 205		SEQ ID NO: 206

miR-148a	miR-148aGSP2	CATGATCAGCTGGGCCAAGAACAAAGTTC	miR-	T+CA+GTGCACTACAGAACT	−0.2928	9.4654	1.27	12.65
		SEQ ID NO: 207	148aRP2	SEQ ID NO: 208

miR-148b	miR-148bGSP2	CATGATCAGCTGGGCCAAGAACAAAGTTC	miR-148bRP	T+CA+GTGCATCACAG	−0.2982	10.417	0.24	2.44
		SEQ ID NO: 209		SEQ ID NO: 210

miR-149	miR-149GSP2	CATGATCAGCTGGGCCAAGAGGAGTGAAG	miR-149RP	T+CT+GGCTCCGTGTC	−0.2996	8.3392	2.15	21.50
		SEQ ID NO: 211		SEQ ID NO: 212

miR-150	miR-150GSP3	CATGATCAGCTGGGCCAAGACACTGGTA	miR-150RP	T+CT+CCCAACCCTTG	−0.2943	8.3945	0.06	0.56
		SEQ ID NO: 213		SEQ ID NO: 214

miR-151	miR-151GSP2	CATGATCAGCTGGGCCAAGACCTCAAGGA	miR-151RP	A+CT+AGACTGAAGCTC	−0.2975	8.651	0.16	1.60
		SEQ ID NO: 215		SEQ ID NO: 216

miR-152	miR-152GSP2	CATGATCAGCTGGGCCAAGACCCAAGTTC	miR-152RP	T+CA+GTGCATGACAG	−0.2741	8.7404	0.33	3.25
		SEQ ID NO: 217		SEQ ID NO: 218

miR-153	miR-153GSP2	CATGATCAGCTGGGCCAAGATCACTTTTG	miR-153RP	TTG+CAT+AGTCACAAAA	−0.2723	9.5732	3.32	33.19
		SEQ ID NO: 219		SEQ ID NO: 220

miR-154*	miR-	CATGATCAGCTGGGCCAAGAAATAGGTCA	miR-	AATCA+TA+CACGGTTGAC	−0.3056	8.8502	0.07	0.74
	154*GSP9^#	SEQ ID NO: 221	154*RP2^#	SEQ ID NO: 222

miR-154	miR-154GSP9^#	CATGATCAGCTGGGCCAAGACGAAGGCAA	miR-	TA+GGTTA+TCCGTGTT	−0.3062	9.3947	0.10	0.96
		SEQ ID NO: 223	154RP3^#	SEQ ID NO: 224

miR-155	miR-155GSP8^#	CATGATCAGCTGGGCCAAGACCCCTATC	miR-	TT+AA+TGCTAATCGTGATAGG	−0.3201	8.474	5.49	54.91
		SEQ ID NO: 225	155RP2^#	SEQ ID NO: 226

miR-181a	miR-	CATGATCAGCTGGGCCAAGAACTCACCGA	miR-	AA+CATT+CAACGCTGTC	−0.2919	7.968	1.70	17.05
	181aGSP9^#	SEQ ID NO: 227	181aRP2^#	SEQ ID NO: 228

miR-181c	miR-	CATGATCAGCTGGGCCAAGAACTCACCGA	miR-	AA+CATT+CAACCTGTCG	−0.3102	7.9029	1.08	10.78
	181cGSP9^#	SEQ ID NO: 229	181cRP2^#	SEQ ID NO: 230

miR-182*	miR-182*GSP	CATGATCAGCTGGGCCAAGATAGTTGGCAA	miR-182*RP	T+GG+TTCTAGACTTGC	−0.2978	8.5876	4.25	42.47
		SEQ ID NO: 231		SEQ ID NO: 232

miR-182	miR-182GSP2	CATGATCAGCTGGGCCAAGATGTGAGTTC	miR-182RP	TTT+GG+CAATGGTAG	−0.2863	9.0854	1.52	15.20
		SEQ ID NO: 233		SEQ ID NO: 234

miR-183	miR-183GSP2	CATGATCAGCTGGGCCAAGACAGTGAATT	miR-183RP	T+AT+GGCACTGGTAG	−0.2774	9.9254	1.95	19.51
		SEQ ID NO: 235		SEQ ID NO: 236

miR-184	miR-184GSP2	CATGATCAGCTGGGCCAAGAACCCTTATC	miR-184RP	T+GG+ACGGAGAACTG	−0.2906	7.9585	0.05	0.49
		SEQ ID NO: 237		SEQ ID NO: 238

miR-186	miR-186GSP9^#	CATGATCAGCTGGGCCAAGAAAGCCCAAA	miR-	CA+AA+GAATT+CTCCTTTTGG	−0.2861	8.6152	0.32	3.18
		SEQ ID NO: 239	186RP3^#	SEQ ID NO: 240

miR-187	miR-187GSP	CATGATCAGCTGGGCCAAGACGGCTGCAAC	miR-187RP	T+CG+TGTCTTGTGTT	−0.2953	7.9329	1.23	12.31
		SEQ ID NO: 241		SEQ ID NO: 242

miR-188	miR-188GSP	CATGATCAGCTGGGCCAAGAACCCTCCACC	miR-188RP	C+AT+CCCTTGCATGG	−0.2925	8.0782	8.49	84.92
		SEQ ID NO: 243		SEQ ID NO: 244

miR-189	miR-189GSP2	CATGATCAGCTGGGCCAAGAACTGATATC	miR-189RP	G+TG+CCTACTGAGCT	−0.2981	8.8964	0.21	2.08
		SEQ ID NO: 245		SEQ ID NO: 246

miR-190	miR-190GSP9^#	CATGATCAGCTGGGCCAAGAACCTAATAT	miR-	T+GA+TA+TGTTTGATATATT	−0.3317	9.8766	0.43	4.34
		SEQ ID NO: 247	190RP4^#	AG
				SEQ ID NO: 248

miR-191	miR-191GSP2	CATGATCAGCTGGGCCAAGAAGCTGCTTT	miR-191RP2	C+AA+CGGAATCCCAAAAG	−0.299	9.0317	0.41	4.07
		SEQ ID NO: 249		SEQ ID NO: 250

miR-192	miR-192GSP2	CATGATCAGCTGGGCCAAGAGGCTGTCAA	miR-192RP	C+TGA+CCTATGAATTGAC	−0.2924	9.5012	1.10	10.98
		SEQ ID NO: 251		SEQ ID NO: 252

miR-193	miR-193GSP9^#	CATGATCAGCTGGGCCAAGACTGGGACTT	miR-	AA+CT+GGCCTACAAAG	−0.3183	8.9942	0.17	1.72
		SEQ ID NO: 253	193RP2^#	SEQ ID NO: 254

miR-194	mir194GSP8^#	CATGATCAGCTGGGCCAAGATCCACATG	mir194RP^#	TG+TAA+CAGCAACTCCA	−0.3078	8.8045	0.37	3.69
		SEQ ID NO: 255		SEQ ID NO: 256

miR-195	miR-195GSP9^#	CATGATCAGCTGGGCCAAGAGCCAATATT	miR-	T+AG+CAG+CACAGAAATA	−0.2955	10.213	0.76	7.58
		SEQ ID NO: 257	195RP3^#	SEQ ID NO: 258

miR-196b	miR-196bGSP	CATGATCAGCTGGGCCAAGACCAACAACAG	miR-196bRP	TA+GGT+AGTTTCCTGT	−0.301	8.1641	1.47	14.66
		SEQ ID NO: 259		SEQ ID NO: 260

miR-196a	miR-196aGSP	CATGATCAGCTGGGCCAAGACCAACAACAT	miR-196aRP	TA+GG+TAGTTTCATGTTG	−0.2932	8.0448	8.04	80.37
		SEQ ID NO: 261		SEQ ID NO: 262

miR-197	miR-197GSP2	CATGATCAGCTGGGCCAAGAGCTGGGTGG	miR-197RP	TT+CA+CCACCTTCTC	−0.289	8.2822	0.71	7.10
		SEQ ID NO: 263		SEQ ID NO: 264

miR-198	miR-198GSP3	CATGATCAGCTGGGCCAAGACCTATCTC	miR-198RP	G+GT+CCAGAGGGGAG	−0.2986	8.1359	0.31	3.15
		SEQ ID NO: 265		SEQ ID NO: 266

miR-	miR-	CATGATCAGCTGGGCCAAGAAACCAATGT	miR-	T+AC+AGTAGTCTGCAC	−0.3029	9.0509	0.25	2.52
199a*	199a*GSP2	SEQ ID NO: 267	199a*RP	SEQ ID NO: 268

miR-199a	miR-199aGSP2	CATGATCAGCTGGGCCAAGAGAACAGGTA	miR-199aRP	C+CC+AGTGTTCAGAC	−0.3187	9.2268	0.12	1.16
		SEQ ID NO: 269		SEQ ID NO: 270

miR-199b	miR-199bGSP	CATGATCAGCTGGGCCAAGAGAACAGATAG	miR-199bRP	C+CC+AGTGTTTAGAC	−0.3165	9.3935	2.00	20.04
		SEQ ID NO: 271		SEQ ID NO: 272

miR-200a	miR-200aGSP2	CATGATCAGCTGGGCCAAGAACATCGTTA	miR-200aRP	TAA+CAC+TGTCTGGT	−0.2754	9.1227	0.08	0.78
		SEQ ID NO: 273		SEQ ID NO: 274

miR-200b	miR-200bGSP2	CATGATCAGCTGGGCCAAGAGTCATCATT	miR-200bRP	TAATA+CTG+CCTGGTAAT	−0.2935	8.5461	0.08	0.85
		SEQ ID NO: 275		SEQ ID NO: 276

miR-202	miR-202	CATGATCAGCTGGGCCAAGATTTTCCCATG	miR-202RP^#	A+GA+GGTATA+GGGCAT	−0.2684	9.056	0.25	2.48
	GSP10^#	SEQ ID NO: 277		SEQ ID NO: 278

miR-203	miR-203GSP2	CATGATCAGCTGGGCCAAGACTAGTGGTC	miR-203RP	G+TG+AAATGTTTAGGACC	−0.2852	8.1279	1.60	16.03
		SEQ ID NO: 279		SEQ ID NO: 280

miR-204	miR-204GSP2	CATGATCAGCTGGGCCAAGAAGGCATAGG	miR-204RP	T+TC+CCTTTGTCATCC	−0.2925	8.7648	0.16	1.59
		SEQ ID NO: 281		SEQ ID NO: 282

miR-205	miR-205GSP	CATGATCAGCTGGGCCAAGACAGACTCCGG	miR-205RP	T+CCTT+CATTCCACC	−0.304	8.2407	9.21	92.15
		SEQ ID NO: 283		SEQ ID NO: 284

miR-206	mir206GSP7^#	CATGATCAGCTGGGCCAAGACCACACA	miR-206RP^#	T+G+GAA+TGTAAGGAAGTGT	−0.2815	8.2206	0.29	2.86
		SEQ ID NO: 285		SEQ ID NO: 286

miR-208	miR-	CATGATCAGCTGGGCCAAGAACAAGCTTTTTGC	miR-	ATAA+GA+CG+AGCAAAAAG	−0.2072	7.9097	57.75	577.52
	208_GSP13^#	SEQ ID NO: 287	208_RP4^#	SEQ ID NO: 288

miR-210	miR-210GSP	CATGATCAGCTGGGCCAAGATCAGCCGCTG	miR-210RP	C+TG+TGCGTGTGACA	−0.2717	8.249	0.18	1.77
		SEQ ID NO: 289		SEQ ID NO: 290

miR-211	miR-211GSP2	CATGATCAGCTGGGCCAAGAAGGCGAAGG	miR-211RP	T+TC+CCTTTGTCATCC	−0.2926	8.3106	0.10	1.00
		SEQ ID NO: 291		SEQ ID NO: 292

miR-212	miR-212GSP9^#	CATGATCAGCTGGGCCAAGAGGCCGTGAC	miR-	T+AA+CAGTCTCCAGTCA	−0.2916	8.0745	0.59	5.86
		SEQ ID NO: 293	212RP2^#	SEQ ID NO: 294

miR-213	miR-213GSP	CATGATCAGCTGGGCCAAGAGGTACAATCA	miR-213RP	A+CC+ATCGACCGTTG	−0.2934	8.1848	2.96	29.59
		SEQ ID NO: 295		SEQ ID NO: 296

miR-214	miR-214GSP	CATGATCAGCTGGGCCAAGACTGCCTGTCT	miR-214RP	A+CA+GCAGGCACAGA	−0.2947	7.82	0.84	8.44
		SEQ ID NO: 297		SEQ ID NO: 298

miR-215	miR-215GSP2	CATGATCAGCTGGGCCAAGAGTCTGTCAA	miR-215RP	A+TGA+CCTATGAATTGAC	−0.2932	8.9273	1.51	15.05
		SEQ ID NO: 299		SEQ ID NO: 300

miR-216	miR-216GSP9^#	CATGATCAGCTGGGCCAAGACACAGTTGC	mir216RP^#	TAA+TCT+CAGCTGGCA	−0.273	8.5829	0.95	9.50
		SEQ ID NO: 301		SEQ ID NO: 302

miR-217	miR-217GSP2	CATGATCAGCTGGGCCAAGAATCCAATCA	miR-217RP2	T+AC+TGCATCAGGAACTGA	−0.3089	9.6502	0.07	0.71
		SEQ ID NO: 303		SEQ ID NO: 304

miR-218	miR-218GSP2	CATGATCAGCTGGGCCAAGAACATGGTTA	miR-218RP	TTG+TGCTT+GATCTAAC	−0.2778	8.4363	1.00	10.05
		SEQ ID NO: 305		SEQ ID NO: 306

miR-220	miR-220GSP	CATGATCAGCTGGGCCAAGAAAAGTGTCAG	miR-220RP	C+CA+CACCGTATCTG	−0.2755	9.0728	8.88	88.75
		SEQ ID NO: 307		SEQ ID NO: 308

miR-221	miR-221GSP9^#	CATGATCAGCTGGGCCAAGAGAAACCCAG	miR-221RP^#	A+GC+TACATTGTCTGC	−0.2886	8.5743	0.12	1.17
		SEQ ID NO: 309		SEQ ID NO: 310

miR-222	miR-222GSP8^#	CATGATCAGCTGGGCCAAGAGAGACCCA	miR-222RP^#	A+GC+TACATCTGGCT	−0.283	8.91	1.64	16.41
		SEQ ID NO: 311		SEQ ID NO: 312

miR-223	miR-223GSP	CATGATCAGCTGGGCCAAGAGGGGTATTTG	miR-223RP	TG+TC+AGTTTGTCAAA	−0.2998	8.6669	0.94	9.44
		SEQ ID NO: 313		SEQ ID NO: 314

miR-224	miR-224GSP8^#	CATGATCAGCTGGGCCAAGATAAACGGA	miR-	C+AAG+TCACTAGTGGTT	−0.2802	7.5575	0.56	5.63
		SEQ ID NO: 315	224RP2^#	SEQ ID NO: 316

miR-296	miR-296GSP9^#	CATGATCAGCTGGGCCAAGAACAGGATTG	miR-	A+GG+GCCCCCCCTCAA	−0.3178	8.3856	0.10	0.96
		SEQ ID NO: 317	296RP2^#	SEQ ID NO: 318

miR-299	miR-299GSP9^#	CATGATCAGCTGGGCCAAGAATGTATGTG	miR-299RP^#	T+GG+TTTACCGTCCC	−0.3155	7.9383	1.30	12.96
		SEQ ID NO: 319		SEQ ID NO: 320

miR-301	miR-301GSP	CATGATCAGCTGGGCCAAGAGCTTTGACAA	miR-301RP	C+AG+TGCAATAGTATTGT	−0.2839	8.314	2.55	25.52
		SEQ ID NO: 321		SEQ ID NO: 322

miR-	miR-302a*GSP	CATGATCAGCTGGGCCAAGAAAAGCAAGTA	miR-	TAAA+CG+TGGATGTAC	−0.2608	8.3921	0.04	0.41
302a*		SEQ ID NO: 323	302a*RP	SEQ ID NO: 324

miR-302a	miR-302aGSP	CATGATCAGCTGGGCCAAGATCACCAAAAC	miR-302aRP	T+AAG+TGCTTCCATGT	−0.2577	9.6657	2.17	21.67
		SEQ ID NO: 325		SEQ ID NO: 326

miR-	miR-302b*GSP	CATGATCAGCTGGGCCAAGAAGAAAGCACT	miR-	A+CTTTAA+CATGGAAGTG	−0.2702	8.5153	0.02	0.24
302b*		SEQ ID NO: 327	302b*RP	SEQ ID NO: 328

miR-302b	miR-302bGSP	CATGATCAGCTGGGCCAAGACTACTAAAAC	miR-302bRP	T+AAG+TGCTTCCATGT	−0.2398	9.1459	5.11	51.11
		SEQ ID NO: 329		SEQ ID NO: 330

miR-302d	miR-302dGSP	CATGATCAGCTGGGCCAAGAACACTCAAAC	miR-302dRP	T+AAG+TGCTTCCATGT	−0.2368	8.5602	5.98	59.78
		SEQ ID NO: 331		SEQ ID NO: 332

miR-	miR-	CATGATCAGCTGGGCCAAGACAGCAGGTA	miR-	TT+TAA+CAT+GGGGGTACC	−0.312	8.2904	0.33	3.28
302c*	302c*_GSP9^#	SEQ ID NO: 333	302c*_RP2^#	SEQ ID NO: 334

miR-302c	miR-	CATGATCAGCTGGGCCAAGACCACTGAAA	miR-	T+AAG+TGCTTCCATGTTTCA	−0.2945	8.381	14.28	142.76
	302cGSP9^#	SEQ ID NO: 335	302cRP5^#	SEQ ID NO: 336

miR-320	miR-	CATGATCAGCTGGGCCAAGATTCGCCCT	miR-	AAAA+GCT+GGGTTGAGAGG	−0.2677	7.8956	6.73	67.29
	320_GSP8^#	SEQ ID NO: 337	320_RP3^#	SEQ ID NO: 338

miR-323	miR-323GSP	CATGATCAGCTGGGCCAAGAAGAGGTCGAC	miR-323RP	G+CA+CATTACACGGT	−0.2878	8.2546	0.19	1.92
		SEQ ID NO: 339		SEQ ID NO: 340

miR-324-	miR-324-	CATGATCAGCTGGGCCAAGACCAGCAGCAC	miR-324-	C+CA+CTGCCCCAGGT	−0.2698	8.5223	2.54	25.41
3p	3pGSP	SEQ ID NO: 341	3pRP	SEQ ID NO: 342

miR-324-	miR-324-	CATGATCAGCTGGGCCAAGAACACCAATGC	miR-324-	C+GC+ATCCCCTAGGG	−0.2861	7.6865	0.06	0.62
5p	5pGSP	SEQ ID NO: 343	5pRP	SEQ ID NO: 344

miR-325	miR-325GSP	CATGATCAGCTGGGCCAAGAACACTTACTG	miR-325RP	C+CT+AGTAGGTGTCC	−0.2976	8.1925	0.01	0.14
		SEQ ID NO: 345		SEQ ID NO: 346

miR-326	miR-326GSP	CATGATCAGCTGGGCCAAGACTGGAGGAAG	miR-326RP	C+CT+CTGGGCCCTTC	−0.2806	7.897	0.59	5.87
		SEQ ID NO: 347		SEQ ID NO: 348

miR-328	miR-328GSP	CATGATCAGCTGGGCCAAGAACGGAAGGGC	miR-328RP	C+TG+GCCCTCTCTGC	−0.293	7.929	3.17	31.69
		SEQ ID NO: 349		SEQ ID NO: 350

miR-330	miR-330GSP	CATGATCAGCTGGGCCAAGATCTCTGCAGG	miR-330RP	G+CA+AAGCACACGGC	−0.3009	7.7999	0.13	1.30
		SEQ ID NO: 351		SEQ ID NO: 352

miR-331	miR-331GSP	CATGATCAGCTGGGCCAAGATTCTAGGATA	miR-331RP	G+CC+CCTGGGCCTAT	−0.2816	8.1643	0.45	4.54
		SEQ ID NO: 353		SEQ ID NO: 354

miR-337	miR-337GSP	CATGATCAGCTGGGCCAAGAAAAGGCATCA	miR-337RP	T+CC+AGCTCCTATATG	−0.2968	8.7313	0.10	1.02
		SEQ ID NO: 355		SEQ ID NO: 356

miR-338	miR-338GSP	CATGATCAGCTGGGCCAAGATCAACAAAAT	miR-338RP2	T+CC+AGCATCAGTGATTT	−0.2768	8.5618	0.52	5.17
		SEQ ID NO: 357		SEQ ID NO: 358

miR-339	miR-339GSP9^#	CATGATCAGCTGGGCCAAGATGAGCTCCT	miR-	T+CC+CTGTCCTCCAGG	−0.303	8.4873	0.27	2.72
		SEQ ID NO: 359	339RP2^#	SEQ ID NO: 360

miR-340	miR-340GSP	CATGATCAGCTGGGCCAAGAGGCTATAAAG	miR-340RP	TC+CG+TCTCAGTTAC	−0.2846	9.6673	0.15	1.45
		SEQ ID NO: 361		SEQ ID NO: 362

miR-342	miR-342GSP3	CATGATCAGCTGGGCCAAGAGACGGGTG	miR-342RP	T+CT+CACACAGAAATCG	−0.293	8.1553	4.69	46.85
		SEQ ID NO: 363		SEQ ID NO: 364

miR-345	miR-345GSP	CATGATCAGCTGGGCCAAGAGCCCTGGACT	miR-345RP	T+GC+TGACTCCTAGT	−0.2909	8.468	0.04	0.40
		SEQ ID NO: 365		SEQ ID NO: 366

miR-346	miR-346GSP	CATGATCAGCTGGGCCAAGAAGAGGCAGGC	miR-346RP	T+GT+CTGCCCGCATG	−0.2959	8.1958	0.25	2.54
		SEQ ID NO: 367		SEQ ID NO: 368

miR-363	miR-363	CATGATCAGCTGGGCCAAGATACAGATGGA	miR-363RP^#	AAT+TG+CAC+GGTATCC	−0.2362	8.9762	0.44	4.36
	GSP10^#	SEQ ID NO: 369		SEQ ID NO: 370

miR-367	miR-367GSP	CATGATCAGCTGGGCCAAGATCACCATTGC	miR-367RP	AAT+TG+CACTTTAGCAAT	−0.2819	8.6711	0.00	0.03
		SEQ ID NO: 371		SEQ ID NO: 372

miR-368	miR-368GSP	CATGATCAGCTGGGCCAAGAAAACGTGGAA	miR-368RP2	A+CATAGA+GGAAATTCCAC	−0.2953	8.0067	6.01	60.11
		SEQ ID NO: 373		SEQ ID NO: 374

miR-370	miR-370GSP	CATGATCAGCTGGGCCAAGACCAGGTTCCA	miR-370RP	G+CC+TGCTGGGGTGG	−0.2825	8.3162	1.45	14.55
		SEQ ID NO: 375		SEQ ID NO: 376

miR-371	miR-371GSP	CATGATCAGCTGGGCCAAGAACACTCAAAA	miR-371RP	G+TG+CCGCCATCTTT	−0.295	7.8812	2.51	25.12
		SEQ ID NO: 377		SEQ ID NO: 378

miR-372	miR-372GSP	CATGATCAGCTGGGCCAAGAACGCTCAAAT	miR-372RP	A+AA+GTGCTGCGACA	−0.2984	8.9183	0.05	0.53
		SEQ ID NO: 379		SEQ ID NO: 380

miR-373*	miR-373*GSP	CATGATCAGCTGGGCCAAGAGGAAAGCGCC	miR-373*RP	A+CT+CAAAATGGGGG	−0.2705	8.4513	0.20	1.99
		SEQ ID NO: 381		SEQ ID NO: 382

miR-373	miR-373GSP	CATGATCAGCTGGGCCAAGAACACCCCAAA	miR-373RP2	GA+AG+TGCTTCGATTTTGG	−0.307	7.9056	9.13	91.32
		SEQ ID NO: 383		SEQ ID NO: 384

miR-374	miR-374GSP2	CATGATCAGCTGGGCCAAGACACTTATCA	miR-374RP	TT+AT+AATA+CAACCTGATA	−0.2655	9.3795	9.16	91.60
		SEQ ID NO: 385		AG
				SEQ ID NO: 386

miR-375	miR-375GSP	CATGATCAGCTGGGCCAAGATCACGCGAGC	miR-375RP	TT+TG+TTCGTTCGGC	−0.3041	8.1181	0.09	0.90
		SEQ ID NO: 387		SEQ ID NO: 388

miR-376b	miR-376b	CATGATCAGCTGGGCCAAGAAACATGGA	miR-	AT+CAT+AGA+GGAAAATCCA	−0.2934	9.0188	1.07	10.74
	GSP8^#	SEQ ID NO: 389	376bRP^#	SEQ ID NO: 390

miR-378	miR-378GSP	CATGATCAGCTGGGCCAAGAACACAGGACC	miR-378RP	C+TC+CTGACTCCAGG	−0.2899	8.1467	0.07	0.73
		SEQ ID NO: 391		SEQ ID NO: 392

miR-379	miR-	CATGATCAGCTGGGCCAAGATACGTTC	miR-	T+GGT+AGACTATGGAACG	−0.2902	8.2149	10.89	108.86
	379_GSP7^#	SEQ ID NO: 393	379RP2^#	SEQ ID NO: 394

miR-380-	miR-380-	CATGATCAGCTGGGCCAAGAGCGCATGTTC	miR-380-	T+GGT+TGACCATAGA	−0.2462	9.4324	1.30	13.04
5p	5pGSP	SEQ ID NO: 395	5pRP	SEQ ID NO: 396

miR-380-	miR-380-	CATGATCAGCTGGGCCAAGAAAGATGTGGA	miR-380-	TA+TG+TAATATGGTCCACA	−0.3037	8.0356	3.69	36.89
3p	3pGSP	SEQ ID NO: 397	3pRP	SEQ ID NO: 398

miR-381	miR-381GSP2	CATGATCAGCTGGGCCAAGAACAGAGAGC	miR-381RP2	TATA+CAA+GGGCAAGCT	−0.3064	8.8704	1.72	17.16
		SEQ ID NO: 399		SEQ ID NO: 400

miR-382	miR-382GSP	CATGATCAGCTGGGCCAAGACGAATCCACC	miR-382RP	G+AA+GTTGTTCGTGGT	−0.2803	7.6738	0.66	6.57
		SEQ ID NO: 401		SEQ ID NO: 402

miR-383	miR-383GSP	CATGATCAGCTGGGCCAAGAAGCCACAATC	miR-383RP2	A+GATC+AGAAGGTGATTGT	−0.2866	8.1463	0.54	5.45
		SEQ ID NO: 403		SEQ ID NO: 404

miR-410	miR-410	CATGATCAGCTGGGCCAAGAACAGGCCAT	miR-410RP^#	AA+TA+TAA+CA+CAGATGGC	−0.2297	8.5166	4.27	42.71
	GSP9^#	SEQ ID NO: 405		SEQ ID NO: 406

miR-412	miR-412	CATGATCAGCTGGGCCAAGAACGGCTAGTG	miR-412RP^#	A+CTT+CACCTGGTCCACTA	−0.3001	7.9099	4.24	42.37
	GSP10^#	SEQ ID NO: 407		SEQ ID NO: 408

miR-422a	miR-422aGSP	CATGATCAGCTGGGCCAAGAGGCCTTCTGA	miR-422aRP	C+TG+GACTTAGGGTC	−0.3079	9.3108	5.95	59.54
		SEQ ID NO: 409		SEQ ID NO: 410

miR-422b	miR-422bGSP	CATGATCAGCTGGGCCAAGAGGCCTTCTGA	miR-422bRP	C+TG+GACTTGGAGTC	−0.2993	8.9437	4.86	48.56
		SEQ ID NO: 411		SEQ ID NO: 412

miR-423	miR-423GSP	CATGATCAGCTGGGCCAAGACTGAGGGGCC	miR-423RP	A+GC+TCGGTCTGAGG	−0.3408	9.2274	6.06	60.62
		SEQ ID NO: 413		SEQ ID NO: 414

miR-424	miR-424GSP^#	CATGATCAGCTGGGCCAAGATTCAAAACAT	miR-	C+AG+CAGCAATTCATGTTTT	−0.3569	9.3419	10.78	107.85
		SEQ ID NO: 415	424RP2^#	SEQ ID NO: 416

miR-425	miR-425GSP	CATGATCAGCTGGGCCAAGAGGCGGACACG	miR-425RP	A+TC+GGGAATGTCGT	−0.2932	7.9786	0.39	3.93
		SEQ ID NO: 417		SEQ ID NO: 418

miR-429	miR-	CATGATCAGCTGGGCCAAGAACGGTTTTACC	miR-	T+AATAC+TG+TCTGGTAAAA	−0.2458	8.2805	16.21	162.12
	429_GSP11^#	SEQ ID NO: 419	429RP5^#	SEQ ID NO: 420

miR-431	miR-431	CATGATCAGCTGGGCCAAGATGCATGACGG	miR-431RP^#	T+GT+CTTGCAGGCCG	−0.3107	7.7127	7.00	70.05
	GSP10^#	SEQ ID NO: 421		SEQ ID NO: 422

miR-448	miR-448GSP	CATGATCAGCTGGGCCAAGAATGGGACATC	miR-448RP	TTG+CATA+TGTAGGATG	−0.3001	8.4969	0.12	1.16
		SEQ ID NO: 423		SEQ ID NO: 424

miR-449	miR-	CATGATCAGCTGGGCCAAGAACCAGCTAAC	miR-	T+GG+CAGTGTATTGTTAGC	−0.3225	8.4953	2.57	25.70
	449GSP10^#	SEQ ID NO: 425	449RP2^#	SEQ ID NO: 426

miR-450	miR-450GSP	CATGATCAGCTGGGCCAAGATATTAGGAAC	miR-450RP	TTTT+TG+CGATGTGTT	−0.2906	8.1404	0.48	4.82
		SEQ ID NO: 427		SEQ ID NO: 428

miR-451	miR-451	CATGATCAGCTGGGCCAAGAAAACTCAGTA	miR-451RP^#	AAA+CCG+TTA+CCATTACTGA	−0.2544	8.0291	1.73	17.35
	GSP10^#	SEQ ID NO: 429		SEQ ID NO: 430

let7a	let7a-GSP2^#	CATGATCAGCTGGGCCAAGAAACTATAC	let7a-RP^#	T+GA+GGTAGTAGGTTG	−0.3089	9.458	0.04	0.38
		SEQ ID NO: 431		SEQ ID NO: 432

let7b	let7b-GSP2^#	CATGATCAGCTGGGCCAAGAAACCACAC	let7b-RP^#	T+GA+GGTAGTAGGTTG	−0.2978	7.9144	0.05	0.54
		SEQ ID NO: 433		SEQ ID NO: 432

let7c	let7c-GSP2^#	CATGATCAGCTGGGCCAAGAAACCATAC	let7c-RP^#	T+GA+GGTAGTAGGTTG	−0.308	7.9854	0.01	0.14
		SEQ ID NO: 434		SEQ ID NO: 432

let7d	let7d-GSP2^#	CATGATCAGCTGGGCCAAGAACTATGCA	let7d-RP^#	A+GA+GGTAGTAGGTTG	−0.3238	8.3359	0.06	0.57
		SEQ ID NO: 435		SEQ ID NO: 436

let7e	let7e-GSP2^#	CATGATCAGCTGGGCCAAGAACTATACA	let7e-RP^#	T+GA+GGTAGGAGGTTG	−0.3284	9.7594	0.22	2.20
		SEQ ID NO: 437		SEQ ID NO: 438

let7f	let7f-GSP2^#	CATGATCAGCTGGGCCAAGAAACTATAC	let7f-RP^#	T+GA+GGTAGTAGATTG	−0.2901	11.107	0.32	3.18
		SEQ ID NO: 439		SEQ ID NO: 440

let7g	let7g-GSP2^#	CATGATCAGCTGGGCCAAGAACTGTACA	let7g-RP^#	T+GA+GGTAGTAGTTTG	−0.3469	9.8235	0.16	1.64
		SEQ ID NO: 441		SEQ ID NO: 442

let7i	let7i-GSP2^#	CATGATCAGCTGGGCCAAGAACAGCACA	let7i-RP^#	T+GA+GGTAGTAGTTTG	−0.321	10.82	0.20	1.99
		SEQ ID NO: 443		SEQ ID NO: 444

miR-377	miR-377GSP	CATGATCAGCTGGGCCAAGAACAAAAGTTG	miR-377RP2	AT+CA+CACAAAGGCAAC	−0.2979	10.612	13.45	134.48
		SEQ ID NO: 445		SEQ ID NO: 446

miR-376a	miR-	CATGATCAGCTGGGCCAAGAACGTGGA	miR-	AT+CAT+AGA+GGAAAATCC	−0.2938	10.045	63.00	630.00
	376a_GSP7	SEQ ID NO: 447	376a_RP5	SEQ ID NO: 448

miR-22	miR-22GSP	CATGATCAGCTGGGCCAAGAACAGTTCTTC	miR-22RP	A+AG+CTGCCAGTTGA	−0.2862	8.883	20.46	204.58
		SEQ ID NO: 449		SEQ ID NO: 450

miR-200c	miR-200cGSP2	CATGATCAGCTGGGCCAAGACCATCATTA	miR-200cRP	T+AA+TACTGCCGGGT	−0.3094	11.5	15.99	159.91
		SEQ ID NO: 451		SEQ ID NO: 452

miR-24	miR-24GSP	CATGATCAGCTGGGCCAAGACTGTTCCTGC	miR-24RP	T+GG+CTCAGTTCAGC	−0.3123	8.6824	24.34	243.38
		SEQ ID NO: 453		SEQ ID NO: 454

miR-	miR-29cGSP10	CATGATCAGCTGGGCCAAGAACCGATTTCA	miR-29cRP	T+AG+CACCATTTGAAAT	−0.2975	8.8441	23.22	232.17
29cDNA		SEQ ID NO: 455		SEQ ID NO: 456

miR-18	miR-18GSP	CATGATCAGCTGGGCCAAGATATCTGCACT	miR-18RP	T+AA+GGTGCATCTAGT	−0.3209	9.0999	14.90	149.01
		SEQ ID NO: 457		SEQ ID NO: 458

miR-185	miR-185GSP	CATGATCAGCTGGGCCAAGAGAACTGCCTT	miR-185RP	T+GG+AGAGAAAGGCA	−0.3081	8.9289	15.73	157.32
		SEQ ID NO: 459		SEQ ID NO: 460

miR-181b	miR-	CATGATCAGCTGGGCCAAGACCCACCGA	miR-	AA+CATT+CATTGCTGTC	−0.3115	10.846	15.87	158.67
	181bGSP8^#	SEQ ID NO: 461	181bRP2^#	SEQ ID NO: 462

miR-128a	miR-128aGSP	CATGATCAGCTGGGCCAAGAAAAAGAGACC	miR-	TCACAGTGAACCGGT	approx.	approx.	approx.	approx.
		SEQ ID NO: 161	128anLRP	SEQ ID NO: 494	−0.2866	8.0867	0.16	1.60

miR-138	miR-138GSP2	CATGATCAGCTGGGCCAAGACGGCCTGAT	miR-	AGCTGGTGTTGTGAA	approx.	approx.	approx.	approx.
		SEQ ID NO: 187	138nLRP	SEQ ID NO: 495	−0.3023	9.0814	0.22	2.19

miR-143	miR-143GSP8^#	CATGATCAGCTGGGCCAAGATGAGCTAC	miR-	TGAGATGAAGCACTGT	approx.	approx.	approx.	approx.
		SEQ ID NO: 197	143nLRP	SEQ ID NO: 496	−0.3008	9.2675	0.37	3.71

miR-150	miR-150GSP3	CATGATCAGCTGGGCCAAGACACTGGTA	miR-	TCTCCCAACCCTTGTA	approx.	approx.	approx.	approx.
		SEQ ID NO: 213	150nLRP	SEQ ID NO: 497	−0.2943	8.3945	0.06	0.56

miR-181a	miR-	CATGATCAGCTGGGCCAAGAACTCACCGA	miR-	AACATTCAACGCTGT	approx.	approx.	approx.	approx.
	181aGSP9^#	SEQ ID NO: 227	181anLRP	SEQ ID NO: 498	−0.2919	7.968	1.70	17.05

miR-194	mir194GSP8^#	CATGATCAGCTGGGCCAAGATCCACATG	miR-	TGTAACAGCAACTCCA	approx.	approx.	approx.	approx.
		SEQ ID NO: 255	194nLRP	SEQ ID NO: 499	−0.3078	8.8045	0.37	3.69

^#denotes primers for assays that required extensive testing and primer design modification to achieve optimal assay results including high sensitivity and high dynamic range.

Example 4

This Example describes assays and primers designed for quantitative analysis of murine miRNA expression patterns.
Methods: The representative murine microRNA target templates described in TABLE 7 are publicly available accessible on the World Wide Web at the Wellcome Trust Sanger Institute website in the “miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al. (2006), Nucleic Acids Research 34: D140-D144. As indicated below in TABLE 7, the murine microRNA templates are either totally identical to the corresponding human microRNA templates, identical in the overlapping sequence with differing ends, or contain one or more base pair changes as compared to the human microRNA sequence. The murine microRNA templates that are identical or that have identical overlapping sequence to the corresponding human templates can be assayed using the same primer sets designed for the human microRNA templates, as indicated in TABLE 7. For the murine microRNA templates with one or more base pair changes in comparison to the corresponding human templates, primer sets have been designed specifically for detection of the murine microRNA, and these primers are provided in TABLE 7. The extension primer reaction and quantitative PCR reactions for detection of the murine microRNA templates may be carried out as described in EXAMPLE 3.

TABLE 7

PRIMERS TO DETECT MURINE MICRORNA TARGET TEMPLATES

Mouse Target					Mouse microRNA as compared
microRNA:	Extension Primer Name	Extension Primer Sequence	Reverse Primer Name	Reverse Primer Sequence	to Human microRNA

miR-1	miR1GSP10	CATGATCAGCTGGGCCAAGATACATACTTC	miR-1RP	T+G+GAA+TG+TAAAGAAGT	Identical
		SEQ ID NO: 47		SEQ ID NO: 48

miR-7	miR-7GSP10	CATGATCAGCTGGGCCAAGAAACAAAATC	miR-7_RP6	T+GGAA+GACTTGTGATTTT	one or more base pairs differ
		SEQ ID NO: 486		SEQ ID NO: 487

miR-9*	miR-9*GSP	CATGATCAGCTGGGCCAAGAACTTTCGGTT	miR-9*RP	TAAA+GCT+AGATAACCG	Identical overlapping sequence,
		SEQ ID NO: 51		SEQ ID NO: 52	ends differ

miR-10a	miR-10aGSP	CATGATCAGCTGGGCCAAGACACAAATTCG	miR-10aRP	T+AC+CCTGTAGATCCG	Identical
		SEQ ID NO: 53		SEQ ID NO: 54

miR-10b	miR-10b_GSP11	CATGATCAGCTGGGCCAAGAACACAAATTCG	miR-10b_RP2	C+CC+TGT+AGAACCGAAT	one or more base pairs differ
		SEQ ID NO: 492		SEQ ID NO: 493

miR-15a	miR-15aGSP	CATGATCAGCTGGGCCAAGACACAAACCAT	miR-15aRP	T+AG+CAGCACATAATG	Identical
		SEQ ID NO: 57		SEQ ID NO: 58

miR-15b	miR-15bGSP2	CATGATCAGCTGGGCCAAGATGTAAACCA	miR-15bRP	T+AG+CAGCACATCAT	Identical
		SEQ ID NO: 59		SEQ ID NO: 60

miR-16	miR-16GSP2	CATGATCAGCTGGGCCAAGACGCCAATAT	miR-16RP	T+AG+CAGCACGTAAA	Identical
		SEQ ID NO: 61		SEQ ID NO: 62

miR-17-3p	miR-17-3pGSP	CATGATCAGCTGGGCCAAGAACAAGTGCCC	miR-17-3pRP	A+CT+GCAGTGAGGGC	one or more base pairs differ
		SEQ ID NO: 463		SEQ ID NO: 464

miR-17-5p	miR-17-5pGSP2	CATGATCAGCTGGGCCAAGAACTACCTGC	miR-17-5pRP	C+AA+AGTGCTTACAGTG	Identical
		SEQ ID NO: 65		SEQ ID NO: 66

miR-19a	miR-19aGSP2	CATGATCAGCTGGGCCAAGATCAGTTTTG	miR-19aRP	TG+TG+CAAATCTATGC	Identical
		SEQ ID NO: 67		SEQ ID NO: 68

miR-19b	miR-19bGSP	CATGATCAGCTGGGCCAAGATCAGTTTTGC	miR-19bRP	TG+TG+CAAATCCATG	Identical
		SEQ ID NO: 69		SEQ ID NO: 70

miR-20	miR-20GSP3	CATGATCAGCTGGGCCAAGACTACCTGC	miR-20RP	T+AA+AGTGCTTATAGTGCA	Identical
		SEQ ID NO: 71		SEQ ID NO: 72

miR-21	miR-21GSP2	CATGATCAGCTGGGCCAAGATCAACATCA	miR-21RP	T+AG+CTTATCAGACTGATG	Identical
		SEQ ID NO: 73		SEQ ID NO: 74

miR-23a	miR-23aGSP	CATGATCAGCTGGGCCAAGAGGAAATCCCT	miR-23aRP	A+TC+ACATTGCCAGG	Identical
		SEQ ID NO: 75		SEQ ID NO: 76

miR-23b	miR-23bGSP	CATGATCAGCTGGGCCAAGAGGTAATCCCT	miR-23bRP	A+TC+ACATTGCCAGG	Identical
		SEQ ID NO: 77		SEQ ID NO: 78

miR-24	miR-24P5	CATGATCAGCTGGGCCAAGACTGTTCCTGC	miR24-1,2R	TGG+CTCAGTTCAGC	Identical
		TG		SEQ ID NO: 19
		SEQ ID NO: 7

miR-25	miR-25GSP	CATGATCAGCTGGGCCAAGATCAGACCGAG	miR-25RP	C+AT+TGCACTTGTCTC	Identical
		SEQ ID NO: 79		SEQ ID NO: 80

miR-26a	miR-26aGSP9	CATGATCAGCTGGGCCAAGAGCCTATCCT	miR-26aRP2	TT+CA+AGTAATCCAGGAT	Identical
		SEQ ID NO: 81		SEQ ID NO: 82

miR-26b	miR-26bGSP9	CATGATCAGCTGGGCCAAGAAACCTATCC	miR-26bRP2	TT+CA+AGT+AATTCAGGAT	Identical
		SEQ ID NO: 83		SEQ ID NO: 84

miR-27a	miR-27aGSP	CATGATCAGCTGGGCCAAGAGCGGAACTTA	miR-27aRP	TT+CA+CAGTGGCTAA	Identical
		SEQ ID NO: 85		SEQ ID NO: 86

miR-27b	miR-27bGSP	CATGATCAGCTGGGCCAAGAGCAGAACTTA	miR-27bRP	TT+CA+CAGTGGCTAA	Identical
		SEQ ID NO: 87		SEQ ID NO: 88

miR-28	miR-28GSP	CATGATCAGCTGGGCCAAGACTCAATAGAC	miR-28RP	A+AG+GAGCTCACAGT	Identical
		SEQ ID NO: 89		SEQ ID NO: 90

miR-29a	miR-29aGSP8	CATGATCAGCTGGGCCAAGAAACCGATT	miR-29aRP2	T+AG+CACCATCTGAAAT	Identical
		SEQ ID NO: 91		SEQ ID NO: 92

miR-29b	miR-29bGSP2	CATGATCAGCTGGGCCAAGAAACACTGAT	miR-29bRP2	T+AG+CACCATTTGAAATCAG	Identical
		SEQ ID NO: 93		SEQ ID NO: 94

miR-30a-5p	miR-30a-5pGSP	CATGATCAGCTGGGCCAAGACTTCCAGTCG	miR-30a-5pRP	T+GT+AAACATCCTCGAC	Identical
		SEQ ID NO: 95		SEQ ID NO: 96

miR-30b	miR-30bGSP	CATGATCAGCTGGGCCAAGAAGCTGAGTGT	miR-30bRP	TGT+AAA+CATCCTACACT	Identical
		SEQ ID NO: 97		SEQ ID NO: 98

miR-30c	miR-30cGSP	CATGATCAGCTGGGCCAAGAGCTGAGAGTG	miR-30cRP	TGT+AAA+CATCCTACACT	Identical
		SEQ ID NO: 99		SEQ ID NO: 100

miR-30d	miR-30dGSP	CATGATCAGCTGGGCCAAGACTTCCAGTCG	miR-30dRP	T+GTAAA+CATCCCCG	Identical
		SEQ ID NO: 101		SEQ ID NO: 102

miR-30e-3p	miR-30e-3pGSP9	CATGATCAGCTGGGCCAAGAGCTGTAAAC	miR-30e-3pRP5	CTTT+CAGT+CGGATGTTT	Identical
		SEQ ID NO: 103		SEQ ID NO: 104

miR-31	miR-31GSP	CATGATCAGCTGGGCCAAGACAGCTATGCC	miR-31RP	G+GC+AAGATGCTGGC	Identical overlapping sequence,
		SEQ ID NO: 107		SEQ ID NO: 108	ends differ

miR-32	miR-32GSP	CATGATCAGCTGGGCCAAGAGCAACTTAGT	miR-32RP	TATTG+CA+CATTACTAAG	Identical
		SEQ ID NO: 109		SEQ ID NO: 110

miR-33	miR-33GSP2	CATGATCAGCTGGGCCAAGACAATGCAAC	miR-33RP	G+TG+CATTGTAGTTGC	Identical
		SEQ ID NO: 111		SEQ ID NO: 112

miR-34a	miR-34aGSP	CATGATCAGCTGGGCCAAGAAACAACCAGC	miR-34aRP	T+GG+CAGTGTCTTAG	Identical
		SEQ ID NO: 113		SEQ ID NO: 114

miR-34b	miR-34bGSP	CATGATCAGCTGGGCCAAGACAATCAGCTA	miR-34bRP	TA+GG+CAGTGTAATT	one or more base pairs differ
		SEQ ID NO: 115		SEQ ID NO: 482

miR-34c	miR-34cGSP	CATGATCAGCTGGGCCAAGAGCAATCAGCT	miR-34cRP	A+GG+CAGTGTAGTTA	Identical
		SEQ ID NO: 117		SEQ ID NO: 118

miR-92	miR-92GSP	CATGATCAGCTGGGCCAAGACAGGCCGGGA	miR-92RP	T+AT+TGCACTTGTCCC	Identical
		SEQ ID NO: 119		SEQ ID NO: 120

miR-93	miR-93GSP	CATGATCAGCTGGGCCAAGACTACCTGCAC	miR-93RP	AA+AG+TGCTGTTCGT	Identical overlapping sequence,
		SEQ ID NO: 121		SEQ ID NO: 122	ends differ

miR-96	miR-96GSP	CATGATCAGCTGGGCCAAGAGCAAAAATGT	miR-96RP	T+TT+GGCACTAGCAC	Identical overlapping sequence,
		SEQ ID NO: 125		SEQ ID NO: 126	ends differ

miR-98	miR-98GSP	CATGATCAGCTGGGCCAAGAAACAATACAA	miR-98RP	TGA+GGT+AGTAAGTTG	Identical
		SEQ ID NO: 127		SEQ ID NO: 128

miR-99a	miR-99aGSP	CATGATCAGCTGGGCCAAGACACAAGATCG	miR-99aRP	A+AC+CCGTAGATCCG	Identical overlapping sequence,
		SEQ ID NO: 129		SEQ ID NO: 130	ends differ

miR-99b	miR-99bGSP	CATGATCAGCTGGGCCAAGACGCAAGGTCG	miR-99bRP	C+AC+CCGTAGAACCG	Identical
		SEQ ID NO: 131		SEQ ID NO: 132

miR-100	miR-100GSP	CATGATCAGCTGGGCCAAGACACAAGTTCG	miR-100RP	A+AC+CCGTAGATCCG	Identical
		SEQ ID NO: 133		SEQ ID NO: 134

miR-101	miR-101GSP	CATGATCAGCTGGGCCAAGACTTCAGTTAT	miR-101RP	TA+CAG+TACTGTGATAACT	Identical
		SEQ ID NO: 135		SEQ ID NO: 136

miR-103	miR-103GSP	CATGATCAGCTGGGCCAAGATCATAGCCCT	miR-103RP	A+GC+AGCATTGTACA	Identical
		SEQ ID NO: 137		SEQ ID NO: 138

miR-106a	miR-106aGSP	CATGATCAGCTGGGCCAAGATACCTGCAC	miR-106aRP	CAA+AG+TGCTAACAGTG	one or more base pairs differ
		SEQ ID NO: 472		SEQ ID NO: 473

miR-106b	miR-106bGSP	CATGATCAGCTGGGCCAAGAATCTGCACTG	miR-106bRP	T+AAAG+TGCTGACAGT	Identical
		SEQ ID NO: 143		SEQ ID NO: 144

miR-107	miR-107GSP8	CATGATCAGCTGGGCCAAGATGATAGCC	miR-107RP2	A+GC+AGCATTGTACAG	Identical
		SEQ ID NO: 145		SEQ ID NO: 146

miR-122a	miR-122aGSP	CATGATCAGCTGGGCCAAGAACAAACACCA	miR-122aRP	T+GG+AGTGTGACAAT	Identical
		SEQ ID NO: 147		SEQ ID NO: 148

miR-124a	miR-124aGSP	CATGATCAGCTGGGCCAAGATGGCATTCAC	miR-124aRP	T+TA+AGGCACGCGGT	Identical overlapping sequence,
		SEQ ID NO: 149		SEQ ID NO: 150	ends differ

miR-125a	miR-125aGSP	CATGATCAGCTGGGCCAAGACACAGGTTAA	miR-125aRP	T+CC+CTGAGACCCTT	Identical
		SEQ ID NO: 151		SEQ ID NO: 152

miR-125b	miR-125bGSP	CATGATCAGCTGGGCCAAGATCACAAGTTA	miR-125bRP	T+CC+CTGAGACCCTA	Identical
		SEQ ID NO: 153		SEQ ID NO: 154

miR-126	miR-126GSP	CATGATCAGCTGGGCCAAGAGCATTATTAC	miR-126RP	T+CG+TACCGTGAGTA	Identical
		SEQ ID NO: 155		SEQ ID NO: 156

miR-126*	miR-126*GSP3	CATGATCAGCTGGGCCAAGACGCGTACC	miR-126*RP	C+ATT+ATTA+CTTTTGGTACG	Identical
		SEQ ID NO: 157		SEQ ID NO: 158

miR-127	miR-127GSP	CATGATCAGCTGGGCCAAGAAGCCAAGCTC	miR-127RP	T+CG+GATCCGTCTGA	Identical overlapping sequence,
		SEQ ID NO: 159		SEQ ID NO: 160	ends differ

miR-128a	miR-128aGSP	CATGATCAGCTGGGCCAAGAAAAAGAGACC	miR-128aRP	T+CA+CAGTGAACCGG	Identical
		SEQ ID NO: 161		SEQ ID NO: 162

miR-128b	miR-128bGSP	CATGATCAGCTGGGCCAAGAGAAAGAGACC	miR-128bRP	T+CA+CAGTGAACCGG	Identical
		SEQ ID NO: 163		SEQ ID NO: 164

miR-130a	miR-130aGSP	CATGATCAGCTGGGCCAAGAATGCCCTTTT	miR-130aRP	C+AG+TGCAATGTTAAAAG	Identical
		SEQ ID NO: 167		SEQ ID NO: 168

miR-130b	miR-130bGSP	CATGATCAGCTGGGCCAAGAATGCCCTTTC	miR-130bRP	C+AG+TGCAATGATGA	Identical
		SEQ ID NO: 169		SEQ ID NO: 170

miR-132	miR-132GSP	CATGATCAGCTGGGCCAAGACGACCATGGC	miR-132RP	T+AA+CAGTCTACAGCC	Identical
		SEQ ID NO: 171		SEQ ID NO: 172

miR-133a	miR-133aGSP	CATGATCAGCTGGGCCAAGAACAGCTGGTT	miR-133aRP	T+TG+GTCCCCTTCAA	Identical
		SEQ ID NO: 173		SEQ ID NO: 174

miR-133b	miR-133bGSP	CATGATCAGCTGGGCCAAGATAGCTGGTTG	miR-133bRP	T+TG+GTCCCCTTCAA	Identical
		SEQ ID NO: 175		SEQ ID NO: 176

miR-134	miR-134GSP	CATGATCAGCTGGGCCAAGACCCTCTGGTC	miR-134RP	T+GT+GACTGGTTGAC	Identical overlapping sequence,
		SEQ ID NO: 177		SEQ ID NO: 178	ends differ

miR-135a	miR-135aGSP	CATGATCAGCTGGGCCAAGATCACATAGGA	miR-135aRP	T+AT+GGCTTTTTATTCCT	Identical
		SEQ ID NO: 179		SEQ ID NO: 180

miR-135b	miR-135bGSP	CATGATCAGCTGGGCCAAGACACATAGGAA	miR-135bRP	T+AT+GGCTTTTCATTCC	Identical
		SEQ ID NO: 181		SEQ ID NO: 182

miR-136	miR-136GSP	CATGATCAGCTGGGCCAAGATCCATCATCA	miR-136RP	A+CT+CCATTTGTTTTGATG	Identical
		SEQ ID NO: 183		SEQ ID NO: 184

miR-137	miR-137GSP	CATGATCAGCTGGGCCAAGACTACGCGTAT	miR-137RP	T+AT+TGCTTAAGAATACGC	Identical overlapping sequence,
		SEQ ID NO: 185		SEQ ID NO: 186	ends differ

miR-138	miR-138GSP2	CATGATCAGCTGGGCCAAGACGGCCTGAT	miR-138RP	A+GC+TGGTGTTGTGA	Identical
		SEQ ID NO: 187		SEQ ID NO: 188

miR-139	miR-139GSP	CATGATCAGCTGGGCCAAGAAGACACGTGC	miR-139RP	T+CT+ACAGTGCACGT	Identical
		SEQ ID NO: 189		SEQ ID NO: 190

miR-140	miR-140GSP	CATGATCAGCTGGGCCAAGACTACCATAGG	miR-140RP	A+GT+GGTTTTACCCT	Identical overlapping sequence,
		SEQ ID NO: 191		SEQ ID NO: 192	ends differ

miR-141	miR-141GSP9	CATGATCAGCTGGGCCAAGACCATCTTTA	miR-141RP2	TAA+CAC+TGTCTGGTAA	Identical
		SEQ ID NO: 193		SEQ ID NO: 194

miR-142-3p	miR-142-3pGSP3	CATGATCAGCTGGGCCAAGATCCATAAA	miR-142-3pRP	TGT+AG+TGTTTCCTACT	Identical overlapping sequence,
		SEQ ID NO: 195		SEQ ID NO: 196	ends differ

miR-143	miR-143GSP8	CATGATCAGCTGGGCCAAGATGAGCTAC	miR-143RP2	T+GA+GATGAAGCACTG	Identical
		SEQ ID NO: 197		SEQ ID NO: 198

miR-144	miR-144GSP2	CATGATCAGCTGGGCCAAGACTAGTACAT	miR-144RP	TA+CA+GTAT+AGATGATG	Identical
		SEQ ID NO: 199		SEQ ID NO: 200

miR-145	miR-145GSP2	CATGATCAGCTGGGCCAAGAAAGGGATTC	miR-145RP	G+TC+CAGTTTTCCCA	Identical
		SEQ ID NO: 201		SEQ ID NO: 202

miR-146	miR-146GSP3	CATGATCAGCTGGGCCAAGAAACCCATG	miR-146RP	T+GA+GAACTGAATTCCA	Identical
		SEQ ID NO: 203		SEQ ID NO: 204

miR-148a	miR-148aGSP2	CATGATCAGCTGGGCCAAGAACAAAGTTC	miR-148aRP2	T+CA+GTGCACTACAGAACT	Identical
		SEQ ID NO: 207		SEQ ID NO: 208

miR-148b	miR-148bGSP2	CATGATCAGCTGGGCCAAGAACAAAGTTC	miR-148bRP	T+CA+GTGCATCACAG	Identical
		SEQ ID NO: 209		SEQ ID NO: 210

miR-149	miR-149GSP2	CATGATCAGCTGGGCCAAGAGGAGTGAAG	miR-149RP	T+CT+GGCTCCGTGTC	Identical
		SEQ ID NO: 211		SEQ ID NO: 212

miR-150	miR-150GSP3	CATGATCAGCTGGGCCAAGACACTGGTA	miR-150RP	T+CT+CCCAACCCTTG	Identical
		SEQ ID NO: 213		SEQ ID NO: 214

miR-151	miR-151GSP2	CATGATCAGCTGGGCCAAGACCTCAAGGA	miR-151RP	A+CT+AGACTGAGGCTC	one or more base pairs differ
		SEQ ID NO: 215		SEQ ID NO: 477

miR-152	miR-152GSP2	CATGATCAGCTGGGCCAAGACCCAAGTTC	miR-152RP	T+CA+GTGCATGACAG	Identical
		SEQ ID NO: 217		SEQ ID NO: 218

miR-153	miR-153GSP2	CATGATCAGCTGGGCCAAGATCACTTTTG	miR-153RP	TTG+CAT+AGTCACAAAA	Identical overlapping sequence,
		SEQ ID NO: 219		SEQ ID NO: 220	ends differ

miR-154	miR-154GSP9	CATGATCAGCTGGGCCAAGACGAAGGCAA	miR-154RP3	TA+GGTTA+TCCGTGTT	Identical
		SEQ ID NO: 223		SEQ ID NO: 224

miR-155	miR-155GSP8	CATGATCAGCTGGGCCAAGACCCCTATC	miR-155RP2	TT+AA+TGCTAATTGTGATAGG	one or more base pairs differ
		SEQ ID NO: 225		SEQ ID NO: 489

miR-181a	miR-181aGSP9	CATGATCAGCTGGGCCAAGAACTCACCGA	miR-181aRP2	AA+CATT+CAACGCTGTC	Identical
		SEQ ID NO: 227		SEQ ID NO: 228

miR-181c	miR-181cGSP9	CATGATCAGCTGGGCCAAGAACTCACCGA	miR-181cRP2	AA+CATT+CAACCTGTCG	Identical
		SEQ ID NO: 229		SEQ ID NO: 230

miR-182	miR-182*GSP	CATGATCAGCTGGGCCAAGATAGTTGGCAA	miR-182*RP	T+GG+TTCTAGACTTGC	Identical
		SEQ ID NO: 231		SEQ ID NO: 232

miR-183	miR-183GSP2	CATGATCAGCTGGGCCAAGACAGTGAATT	miR-183RP	T+AT+GGCACTGGTAG	Identical
		SEQ ID NO: 235		SEQ ID NO: 236

miR-184	miR-184GSP2	CATGATCAGCTGGGCCAAGAACCCTTATC	miR-184RP	T+GG+ACGGAGAACTG	Identical
		SEQ ID NO: 237		SEQ ID NO: 238

miR-186	miR-186GSP9	CATGATCAGCTGGGCCAAGAAAGCCCAAA	miR-186RP3	CA+AA+GAATT+CTCCTTTTGG	Identical
		SEQ ID NO: 239		SEQ ID NO: 240

miR-187	miR-187GSP	CATGATCAGCTGGGCCAAGACGGCTGCAAC	miR-187RP	T+CG+TGTCTTGTGTT	Identical overlapping sequence,
		SEQ ID NO: 241		SEQ ID NO: 242	ends differ

miR-188	miR-188GSP	CATGATCAGCTGGGCCAAGAACCCTCCACC	miR-188RP	C+AT+CCCTTGCATGG	Identical
		SEQ ID NO: 243		SEQ ID NO: 244

miR-189	miR-189GSP2	CATGATCAGCTGGGCCAAGAACTGATATC	miR-189RP	G+TG+CCTACTGAGCT	Identical
		SEQ ID NO: 245		SEQ ID NO: 246

miR-190	miR-190GSP9	CATGATCAGCTGGGCCAAGAACCTAATAT	miR-190RP4	T+GA+TA+TGTTTGATATATTAG	Identical
		SEQ ID NO: 247		SEQ ID NO: 248

miR-191	miR-191GSP2	CATGATCAGCTGGGCCAAGAAGCTGCTTT	miR-191RP2	C+AA+CGGAATCCCAAAAG	Identical
		SEQ ID NO: 249		SEQ ID NO: 250

miR-192	miR-192GSP2	CATGATCAGCTGGGCCAAGAGGCTGTCAA	miR-192RP	C+TGA+CCTATGAATTGAC	Identical overlapping sequence,
		SEQ ID NO: 251		SEQ ID NO: 252	ends differ

miR-193	miR-193GSP9	CATGATCAGCTGGGCCAAGACTGGGACTT	miR-193RP2	AA+CT+GGCCTACAAAG	Identical
		SEQ ID NO: 253		SEQ ID NO: 254

miR-194	mir194GSP8	CATGATCAGCTGGGCCAAGATCCACATG	mir194RP	TG+TAA+CAGCAACTCCA	Identical
		SEQ ID NO: 255		SEQ ID NO: 256

miR-195	miR-195GSP9	CATGATCAGCTGGGCCAAGAGCCAATATT	miR-195RP3	T+AG+CAG+CACAGAAATA	Identical
		SEQ ID NO: 257		SEQ ID NO: 258

miR-196a	miR-196aGSP	CATGATCAGCTGGGCCAAGACCAACAACAT	miR-196aRP	TA+GG+TAGTTTCATGTTG	Identical
		SEQ ID NO: 261		SEQ ID NO: 262

miR-196b	miR-196bGSP	CATGATCAGCTGGGCCAAGACCAACAACAG	miR-196bRP	TA+GGT+AGTTTCCTGT	Identical
		SEQ ID NO: 259		SEQ ID NO: 260

miR-199a*	miR-199a*GSP2	CATGATCAGCTGGGCCAAGAAACCAATGT	miR-199a*RP	T+AC+AGTAGTCTGCAC	Identical
		SEQ ID NO: 267		SEQ ID NO: 268

miR-199a	miR-199aGSP2	CATGATCAGCTGGGCCAAGAGAACAGGTA	miR-199aRP	C+CC+AGTGTTCAGAC	Identical
		SEQ ID NO: 269		SEQ ID NO: 270

miR-199b	miR-199bGSP	CATGATCAGCTGGGCCAAGAGAACAGGTAG	miR-199bRP	C+CC+AGTGTTTAGAC	one or more base pairs differ
		SEQ ID NO: 475		SEQ ID NO: 272

miR-200a	miR-200aGSP2	CATGATCAGCTGGGCCAAGAACATCGTTA	miR-200aRP	TAA+CAC+TGTCTGGT	Identical
		SEQ ID NO: 273		SEQ ID NO: 274

miR-200b	miR-200bGSP2	CATGATCAGCTGGGCCAAGAGTCATCATT	miR-200bRP	TAATA+CTG+CCTGGTAAT	Identical
		SEQ ID NO: 275		SEQ ID NO: 276

miR-203	miR-203GSP2	CATGATCAGCTGGGCCAAGACTAGTGGTC	miR-203RP	G+TG+AAATGTTTAGGACC	Identical overlapping sequence,
		SEQ ID NO: 279		SEQ ID NO: 280	ends differ

miR-204	miR-204GSP2	CATGATCAGCTGGGCCAAGAAGGCATAGG	miR-204RP	T+TC+CCTTTGTCATCC	Identical overlapping sequence,
		SEQ ID NO: 281		SEQ ID NO: 282	ends differ

miR-205	miR-205GSP	CATGATCAGCTGGGCCAAGACAGACTCCGG	miR-205RP	T+CCTT+CATTCCACC	Identical
		SEQ ID NO: 283		SEQ ID NO: 284

miR-206	mir206GSP7	CATGATCAGCTGGGCCAAGACCACACA	miR-206RP	T+G+GAA+TGTAAGGAAGTGT	Identical
		SEQ ID NO: 285		SEQ ID NO: 286

miR-208	miR-208_GSP13	CATGATCAGCTGGGCCAAGAACAAGCTTTT	miR-208_RP4	ATAA+GA+CG+AGCAAAAAG	Identical
		TGC		SEQ ID NO: 288
		SEQ ID NO: 287

miR-210	miR-210GSP	CATGATCAGCTGGGCCAAGATCAGCCGCTG	miR-210RP	C+TG+TGCGTGTGACA	Identical
		SEQ ID NO: 289		SEQ ID NO: 290

miR-211	miR-211GSP2	CATGATCAGCTGGGCCAAGAAGGCAAAGG	miR-211RP	T+TC+CCTTTGTCATCC	one or more base pairs differ
		SEQ ID NO: 491		SEQ ID NO: 292

miR-212	miR-212GSP9	CATGATCAGCTGGGCCAAGAGGCCGTGAC	miR-212RP2	T+AA+CAGTCTCCAGTCA	Identical
		SEQ ID NO: 293		SEQ ID NO: 294

miR-213	miR-213GSP	CATGATCAGCTGGGCCAAGAGGTACAATCA	miR-213RP	A+CC+ATCGACCGTTG	Identical
		SEQ ID NO: 295		SEQ ID NO: 296

miR-214	miR-214GSP	CATGATCAGCTGGGCCAAGACTGCCTGTCT	miR-214RP	A+CA+GCAGGCACAGA	Identical
		SEQ ID NO: 297		SEQ ID NO: 298

miR-215	miR-215GSP2	CATGATCAGCTGGGCCAAGAGTCTGTCAA	miR-215RP	A+TGA+CCTATGATTTGAC	one or more base pairs differ
		SEQ ID NO: 299		SEQ ID NO: 469

miR-216	miR-216GSP9	CATGATCAGCTGGGCCAAGACACAGTTGC	mir216RP	TAA+TCT+CAGCTGGCA	Identical
		SEQ ID NO: 301		SEQ ID NO: 302

miR-217	miR-217GSP2	CATGATCAGCTGGGCCAAGAATCCAGTCA	miR-217RP2	T+AC+TGCATCAGGAACTGA	one or more base pairs differ
		SEQ ID NO: 481		SEQ ID NO: 304

miR-218	miR-218GSP2	CATGATCAGCTGGGCCAAGAACATGGTTA	miR-218RP	TTG+TGCTT+GATCTAAC	Identical
		SEQ ID NO: 305		SEQ ID NO: 306

miR-221	miR-221GSP9	CATGATCAGCTGGGCCAAGAGAAACCCAG	miR-221RP	A+GC+TACATTGTCTGC	Identical overlapping sequence,
		SEQ ID NO: 309		SEQ ID NO: 310	ends differ

miR-222	miR-222GSP8	CATGATCAGCTGGGCCAAGAGAGACCCA	miR-222RP	A+GC+TACATCTGGCT	Identical
		SEQ ID NO: 311		SEQ ID NO: 312

miR-223	miR-223GSP	CATGATCAGCTGGGCCAAGAGGGGTATTTG	miR-223RP	TG+TC+AGTTTGTCAAA	Identical
		SEQ ID NO: 313		SEQ ID NO: 314

miR-224	miR-224GSP8	CATGATCAGCTGGGCCAAGATAAACGGA	miR-224RP2	C+AAG+TCACTAGTGGTT	Identical overlapping sequence,
		SEQ ID NO: 315		SEQ ID NO: 316	ends differ

miR-296	miR-296GSP9	CATGATCAGCTGGGCCAAGAACAGGATTG	miR-296RP2	A+GG+GCCCCCCCTCAA	Identical
		SEQ ID NO: 317		SEQ ID NO: 318

miR-299	miR-299GSP9	CATGATCAGCTGGGCCAAGAATGTATGTG	miR-299RP	T+GG+TTTACCGTCCC	Identical
		SEQ ID NO: 319		SEQ ID NO: 320

miR-301	miR-301GSP	CATGATCAGCTGGGCCAAGAGCTTTGACAA	miR-301RP	C+AG+TGCAATAGTATTGT	Identical
		SEQ ID NO: 321		SEQ ID NO: 322

miR-302a	miR-302aGSP	CATGATCAGCTGGGCCAAGATCACCAAAAC	miR-302aRP	T+AAG+TGCTTCCATGT	Identical
		SEQ ID NO: 325		SEQ ID NO: 326

miR-320	miR-320_GSP8	CATGATCAGCTGGGCCAAGATTCGCCCT	miR-320_RP3	AAAA+GCT+GGGTTGAGAGG	Identical
		SEQ ID NO: 337		SEQ ID NO: 338

miR-323	miR-323GSP	CATGATCAGCTGGGCCAAGAAGAGGTCGAC	miR-323RP	G+CA+CATTACACGGT	Identical
		SEQ ID NO: 339		SEQ ID NO: 340

miR-324-3p	miR-324-3pGSP	CATGATCAGCTGGGCCAAGACCAGCAGCAC	miR-324-3pRP	C+CA+CTGCCCCAGGT	Identical
		SEQ ID NO: 341		SEQ ID NO: 342

miR-324-5p	miR-324-5pGSP	CATGATCAGCTGGGCCAAGAACACCAATGC	miR-324-5pRP	C+GC+ATCCCCTAGGG	Identical overlapping sequence,
		SEQ ID NO: 343		SEQ ID NO: 344	ends differ

miR-325	miR-325GSP	CATGATCAGCTGGGCCAAGAACACTTACTG	miR-325RP	C+CT+AGTAGGTGCTC	one or more base pairs differ
		SEQ ID NO: 345		SEQ ID NO: 476

miR-326	miR-326GSP	CATGATCAGCTGGGCCAAGACTGGAGGAAG	miR-326RP	C+CT+CTGGGCCCTTC	Identical overlapping sequence,
		SEQ ID NO: 347		SEQ ID NO: 348	ends differ

miR-328	miR-328GSP	CATGATCAGCTGGGCCAAGAACGGAAGGGC	miR-328RP	C+TG+GCCCTCTCTGC	Identical
		SEQ ID NO: 349		SEQ ID NO: 350

miR-330	miR-330GSP	CATGATCAGCTGGGCCAAGATCTCTGCAGG	miR-330RP	G+CA+AAGCACAGGGC	one or more base pairs differ
		SEQ ID NO: 351		SEQ ID NO: 478

miR-331	miR-331GSP	CATGATCAGCTGGGCCAAGATTCTAGGATA	miR-331RP	G+CC+CCTGGGCCTAT	Identical
		SEQ ID NO: 353		SEQ ID NO: 354

miR-337	miR-337GSP	CATGATCAGCTGGGCCAAGAAAAGGCATCA	miR-337RP	T+TC+AGCTCCTATATG	one or more base pairs differ
		SEQ ID NO: 355		SEQ ID NO: 490

miR-338	miR-338GSP	CATGATCAGCTGGGCCAAGATCAACAAAAT	miR-338RP2	T+CC+AGCATCAGTGATTT	Identical
		SEQ ID NO: 357		SEQ ID NO: 358

miR-339	miR-339GSP9	CATGATCAGCTGGGCCAAGATGAGCTCCT	miR-339RP2	T+CC+CTGTCCTCCAGG	Identical
		SEQ ID NO: 359		SEQ ID NO: 360

miR-340	miR-340GSP	CATGATCAGCTGGGCCAAGAGGCTATAAAG	miR-340RP	TC+CG+TCTCAGTTAC	Identical
		SEQ ID NO: 361		SEQ ID NO: 362

miR-342	miR-342GSP3	CATGATCAGCTGGGCCAAGAGACGGGTG	miR-342RP	T+CT+CACACAGAAATCG	Identical
		SEQ ID NO: 363		SEQ ID NO: 364

miR-345	miR-345GSP	CATGATCAGCTGGGCCAAGAGCACTGGACT	miR-345RP	T+GC+TGACCCCTAGT	one or more base pairs differ
		SEQ ID NO: 484		SEQ ID NO: 485

miR-346	miR-346GSP	CATGATCAGCTGGGCCAAGAAGAGGCAGGC	miR-346RP	T+GT+CTGCCCGAGTG	one or more base pairs differ
		SEQ ID NO: 367		SEQ ID NO: 488

miR-363	miR-363 GSP10	CATGATCAGCTGGGCCAAGATACAGATGGA	miR-363RP	AAT+TG+CAC+GGTATCC	Identical
		SEQ ID NO: 369		SEQ ID NO: 370

miR-370	miR-370GSP	CATGATCAGCTGGGCCAAGACCAGGTTCCA	miR-370RP	G+CC+TGCTGGGGTGG	Identical overlapping sequence,
		SEQ ID NO: 375		SEQ ID NO: 376	ends differ

miR-375	miR-375GSP	CATGATCAGCTGGGCCAAGATCACGCGAGC	miR-375RP	TT+TG+TTCGTTCGGC	Identical
		SEQ ID NO: 387		SEQ ID NO: 388

miR-376a	miR-376aGSP3	CATGATCAGCTGGGCCAAGAACGTGGAT	miR-376aRP2	A+TCGTAGA+GGAAAATCCAC	one or more base pairs differ
		SEQ ID NO: 467		SEQ ID NO: 468

miR-378	miR-378GSP	CATGATCAGCTGGGCCAAGAACACAGGACC	miR-378RP	C+TC+CTGACTCCAGG	Identical
		SEQ ID NO: 391		SEQ ID NO: 392

miR-379	miR-379_GSP7	CATGATCAGCTGGGCCAAGATACGTTC	miR-379RP2	T+GGT+AGACTATGGAACG	Identical overlapping sequence,
		SEQ ID NO: 393		SEQ ID NO: 394	ends differ

miR-380-5p	miR-380-5pGSP	CATGATCAGCTGGGCCAAGAGCGCATGTTC	miR-380-5pRP	T+GGT+TGACCATAGA	Identical
		SEQ ID NO: 395		SEQ ID NO: 396

miR-380-3p	miR-380-3pGSP	CATGATCAGCTGGGCCAAGAAAGATGTGGA	miR-380-3pRP	TA+TG+TAGTATGGTCCACA	one or more base pairs differ
		SEQ ID NO: 395		SEQ ID NO: 483

miR-381	miR-381GSP2	CATGATCAGCTGGGCCAAGAACAGAGAGC	miR-381RP2	TATA+CAA+GGGCAAGCT	Identical
		SEQ ID NO: 399		SEQ ID NO: 400

miR-382	miR-382GSP	CATGATCAGCTGGGCCAAGACGAATCCACC	miR-382RP	G+AA+GTTGTTCGTGGT	Identical
		SEQ ID NO: 401		SEQ ID NO: 402

miR-383	miR-383GSP	CATGATCAGCTGGGCCAAGAAGCCACAGTC	miR-383RP2	A+GATC+AGAAGGTGACTGT	one or more base pairs differ
		SEQ ID NO: 465		SEQ ID NO: 466

miR-384	miR-384_GSP9	CATGATCAGCTGGGCCAAGATGTGAACAA	miR-384_RP5	ATT+CCT+AG+AAATTGTTC	one or more base pairs differ
		SEQ ID NO: 470		SEQ ID NO: 471

miR-410	miR-410 GSP9	CATGATCAGCTGGGCCAAGAACAGGCCAT	miR-410RP	AA+TA+TAA+CA+CAGATGGC	Identical
		SEQ ID NO: 405		SEQ ID NO: 406

miR-412	miR-412 GSP10	CATGATCAGCTGGGCCAAGAACGGCTAGTG	miR-412RP	A+CTT+CACCTGGTCCACTA	Identical
		SEQ ID NO: 407		SEQ ID NO: 408

miR-424	miR-424GSP	CATGATCAGCTGGGCCAAGATCCAAAACAT	miR-424RP2	C+AG+CAGCAATTCATGTTTT	one or more base pairs differ
		SEQ ID NO: 474		SEQ ID NO: 414

miR-425	miR-425GSP	CATGATCAGCTGGGCCAAGAGGCGGACACG	miR-425RP	A+TC+GGGAATGTCGT	Identical
		SEQ ID NO: 417		SEQ ID NO: 418

miR-429	miR-429_GSP11	CATGATCAGCTGGGCCAAGAACGGCATTACC	miR-429RP5	T+AATAC+TG+TCTGGTAATG	one or more base pairs differ
		SEQ ID NO: 479		SEQ ID NO: 480

miR-431	miR-431 GSP10	CATGATCAGCTGGGCCAAGATGCATGACGG	miR-431RP	T+GT+CTTGCAGGCCG	Identical overlapping sequence,
		SEQ ID NO: 421		SEQ ID NO: 422	ends differ

miR-448	miR-448GSP	CATGATCAGCTGGGCCAAGAATGGGACATC	miR-448RP	TTG+CATA+TGTAGGATG	Identical
		SEQ ID NO: 423		SEQ ID NO: 424

miR-449	miR-449GSP10	CATGATCAGCTGGGCCAAGAACCAGCTAAC	miR-449RP2	T+GG+CAGTGTATTGTTAGC	Identical
		SEQ ID NO: 425		SEQ ID NO: 426

miR-450	miR-450GSP	CATGATCAGCTGGGCCAAGATATTAGGAAC	miR-450RP	TTTT+TG+CGATGTGTT	Identical
		SEQ ID NO: 427		SEQ ID NO: 428

miR-451	miR-451 GSP10	CATGATCAGCTGGGCCAAGAAAACTCAGTA	miR-451RP	AAA+CCG+TTA+CCATTACTGA	Identical overlapping sequence,
		SEQ ID NO: 429		SEQ ID NO: 430	ends differ

let7a	let7a-GSP2	CATGATCAGCTGGGCCAAGAAACTATAC	let7a-RP	T+GA+GGTAGTAGGTTG	Identical overlapping sequence,
		SEQ ID NO: 431		SEQ ID NO: 432	ends differ

let7b	let7b-GSP2	CATGATCAGCTGGGCCAAGAAACCACAC	let7b-RP	T+GA+GGTAGTAGGTTG	Identical
		SEQ ID NO: 433		SEQ ID NO: 432

let7c	let7c-GSP2	CATGATCAGCTGGGCCAAGAAACCATAC	let7c-RP	T+GA+GGTAGTAGGTTG	Identical
		SEQ ID NO: 434		SEQ ID NO: 432

let7d	let7d-GSP2	CATGATCAGCTGGGCCAAGAACTATGCA	let7d-RP	A+GA+GGTAGTAGGTTG	Identical
		SEQ ID NO: 435		SEQ ID NO: 436

let7e	let7e-GSP2	CATGATCAGCTGGGCCAAGAACTATACA	let7e-RP	T+GA+GGTAGGAGGTTG	Identical
		SEQ ID NO: 437		SEQ ID NO: 438

let7f	let7f-GSP2	CATGATCAGCTGGGCCAAGAAACTATAC	let7f-RP	T+GA+GGTAGTAGATTG	Identical overlapping sequence,
		SEQ ID NO: 439		SEQ ID NO: 440	ends differ

let7g	let7g-GSP2	CATGATCAGCTGGGCCAAGAACTGTACA	let7g-RP	T+GA+GGTAGTAGTTTG	Identical
		SEQ ID NO: 441		SEQ ID NO: 442

let7i	let7i-GSP2	CATGATCAGCTGGGCCAAGAACAGCACA	let7i-RP	T+GA+GGTAGTAGTTTG	Identical
		SEQ ID NO: 443		SEQ ID NO: 444

Example 5

This Example describes the detection and analysis of expression profiles for three microRNAs in total RNA isolated from twelve different tissues using methods in accordance with an embodiment of the present invention.
Methods: Quantitative analysis of miR-1, miR-124 and miR-150 microRNA templates was determined using 0.5 μg of First Choice total RNA (Ambion, Inc.) per 10 μl primer extension reaction isolated from the following tissues: brain, heart, intestine, kidney, liver, lung, lymph, ovary, skeletal muscle, spleen, thymus and uterus. The primer extension enzyme and quantitative PCR reactions were carried out as described above in EXAMPLE 3, using the following PCR primers:
miR-1 Template:
extension primer:

(SEQ ID NO: 47)

CATGATCAGCTGGGCCAAGATACATACTTC

reverse primer:

(SEQ ID NO: 48)

T+G+GAA+TG+TAAAGAAGT

forward primer:

(SEQ ID NO: 13)

CATGATCAGCTGGGCCAAGA

miR-124 Template:
extension primer:

(SEQ ID NO: 149)

CATGATCAGCTGGGCCAAGATGGCATTCAC

reverse primer:

(SEQ ID NO: 150)

T+TA+AGGCACGCGGT

forward primer:

(SEQ ID NO: 13)

CATGATCAGCTGGGCCAAGA

miR-150 template:

extension primer:

(SEQ ID NO: 213)

	CATGATCAGCTGGGCCAAGACACTGGTA

	reverse primer:

(SEQ ID NO: 214)

	T+CT+CCCAACCCTTG

	forward primer:

(SEQ ID NO: 13)

CATGATCAGCTGGGCCAAGA

Results. The expression profiles for miR-1, miR-124 and miR-150 are shown in FIGS. 3A, 3B, and 3C, respectively. The data in FIGS. 3A-3C are presented in units of microRNA copies per 10 pg of total RNA (y-axis). These units were chosen since human cell lines typically yield ≦10 pg of total RNA per cell. Hence the data shown are estimates of microRNA copies per cell. The numbers on the x-axis correspond to the following tissues: (1) brain, (2) heart, (3) intestine, (4) kidney, (5) liver, (6) lung, (7) lymph, (8) ovary, (9) skeletal muscle, (10) spleen, (11) thymus and (12) uterus.
Consistent with previous reports, very high levels of striated muscle-specific expression were found for miR-1 (as shown in FIG. 3A), and high levels of brain expression were found for miR-124 (as shown in FIG. 3B) (see Lagos-Quintana et al., RNA 9:175-179, 2003). Quantitative analysis reveals that these microRNAs are present at tens to hundreds of thousands of copies per cell. These data are in agreement with quantitative Northern blot estimates of miR-1 and miR-124 levels (see Lim et al., Nature 433:769-773, 2005). As shown in FIG. 3C, miR-150 was found to be highly expressed in the immune-related lymph node, thymus and spleen samples which is also consistent with previous findings (see Baskerville et al., RNA 11:241-247, 2005).

Example 6

This Example describes the selection and validation of primers for detecting mammalian microRNAs of interest.
Rationale: In order to perform multiple assays to detect a plurality of microRNA targets in a single sample (i.e., multiplex PCR), it is important that the assays work under uniform reverse transcriptase and PCR cycling conditions in a common buffer system with a single universal primer. The following primer design principles and high throughput assays were utilized to identify useful primer sets for desired microRNA targets that work well under the designated reaction conditions.
Primer Design:
As described in Example 2, the sensitivity of an assay to detect mammalian microRNA targets using the methods of the invention may be measured by the cycle threshold (Ct) value. The lower the Ct value (e.g., the fewer number of cycles), the more sensitive is the assay. The ΔCt value is the difference between the number of cycles (Ct) between template containing samples and no template controls, and serves as a measure of the dynamic range of the assay. Assays with a high dynamic range allow measurements of very low microRNA copy numbers. Accordingly, desirable characteristics of a microRNA detection assay include high sensitivity (low Ct value) (preferably in the range of from about 5 to about 25, such as from about 10 to about 20), and broad dynamic range (preferably in the range of from about 10 and 35, such as ΔCt≧12) between the signal of a sample containing target template and a no template background control sample.
microRNA Target Templates: Representative mammalian microRNA target templates (h=human, r=rat, m=mouse) are provided in Table 9 (SEQ ID NO:966 to SEQ ID NO:1043) which are publicly available and accessible on the World Wide Web at the Wellcome Trust Sanger Institute website in the “miRBase sequence database” as described in Griffith-Jones et al. (2004), Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al. (2006), Nucleic Acids Research 34:D140-D144.
Extension Primers:
Empirical data generated as described in Examples 1-5 suggests that gene specific (GS) extension primers are primarily responsible for the dynamic range of the assays for detecting mammalian microRNA targets using the methods described herein. As described in Example 2, it was determined that the dynamic range (ΔCt) and specificity of the assays tested decreased for extension primers having gene specific regions below 6 to 7 nucleotides. Therefore, in order to optimize microRNA detection assays, extension primers were designed that have 7 to 10 nucleotide overlap with the microRNA target of interest. Exemplary extension primers for the microRNA targets listed in TABLE 9 are provided in TABLE 8 (SEQ ID NO:500 to SEQ ID NO:965). These exemplary extension primers have a gene specific (GS) region from 7 to 10 nucleotide overlap with the microRNA target of interest.
Reverse Primers:
Unmodified and locked nucleic acid (LNA)-containing reverse primers were designed to quantify the primer-extended, full length cDNA in combination with a generic universal forward primer (SEQ ID NO:13). Based on the data generated as described in Examples 1-5, it was determined that the design of the reverse primers contributes to the efficiency of the PCR reactions, with the observation that the longer the reverse primer, the better the PCR performance. However, it was also observed that the longer the overlap with the extension primer, the higher the background. Therefore, the reverse primers were designed to be as long as possible while minimizing the overlap with the gene specific portion of the extension primer, in order to reduce the non-specific background signal.
In addition, as described in Example 3, LNA base substitutions may be selected to raise the predicted Tm of the primer, with two or three LNA base substitutions typically substituted within the first 8 nucleotides from the 5′ end of the reverse primer oligonucleotide. Exemplary reverse primers for the microRNA targets listed in TABLE 9 are provided in TABLE 8. While these exemplary reverse primers contain LNA base substitutions (the “+” symbol preceding a nucleotide designates an LNA substitution), this feature is optional and not required.
Selection and validation of primers for a desired target:
Assay oligonucleotide selection is made in two steps as follows:
1) Primer designs were determined using the principles described above. Typically, 4 extension primer candidates and 2 reverse primer candidates were designed for each microRNA target of interest. The extension primers in each set overlap the gene specific region by 7, 8, 9 and 10 nucleotides, respectively, at the 3′ end. Exemplary primers designed according to these design principles are provided in TABLE 8 for the microRNA targets listed in TABLE 9.
Assay design to validate the candidate primer sets (Assay #1)
microRNA Target:
Exemplary target microRNA miR-495 has an RNA target sequence (SEQ ID NO:966) that is conserved across human (h), mouse (m) and rat (r), as indicated by the designation “hmr”-miR-495 in TABLE 9. Therefore, the primer designed for this target sequence would be expected to be useful to detect miR-495 in samples obtained from human, mouse, and rat.
microRNA miR-495 target RNA sequence: 5′ AAACAAACAUGGUGCACUUCUU 3′ (SEQ ID NO:966)

Extension Primers (4 candidates)

(SEQ ID NO: 500)

hmr-miR-495GS10: 5′ CATGATCAGCTGGGCCAAGAAAGAAGTGCA

3′

(SEQ ID NO: 501)

hmr-miR-495GS9: 5′ CATGATCAGCTGGGCCAAGAAAGAAGTGC

3′

(SEQ ID NO: 502)

hmr-miR-495GS8: 5′ CATGATCAGCTGGGCCAAGAAAGAAGTG 3′

(SEQ ID NO: 503)

hmr-miR-495GS7: 5′ CATGATCAGCTGGGCCAAGAAAGAAGT

Reverse Primers (2 candidates)

(SEQ ID NO: 504)

hmr-miR-495RP1: 5′ AAA+CAAA+CA+TGGTGCAC 3′

(SEQ ID NO: 505)

hmr-miR-495RP2: 5′ AAA+C+AAA+CATGGTGC 3′

2) The primers designed as described above were tested to find pairs that showed both high sensitivity and high dynamic range in quantitative PCR assays, using the assay methods described in Example 2. The optimal combination of extension primer and reverse primer was determined for the target microRNA by testing all combinations of primers in the presence or absence of DNA template. It is preferable to use DNA rather than RNA template to test the oligo pairs because it is less likely to degrade than RNA. Degraded templates result in misleading assay data. Therefore, HPLC purified DNA template molecules are preferred.
TABLE 8 shows exemplary primer sets for use in detection assays for 78 microRNA targets (shown in TABLE 9). The candidate primers for use in these assays were designed to specifically detect human (h), mouse (m) and rat (r) microRNAs, or microRNAs from one or more species. For example, assays with the “hmr” prefix are designed to detect a perfectly conserved microRNA in all three species, whereas a “mr” prefix means the assay is designed to detect a microRNA conserved between mouse and rat, but not human. Nucleotides preceded by a plus (+) sign may be optionally locked (LNA). TABLE 9 shows the microRNA target sequence for each assay.

TABLE 8

EXEMPLARY PRIMER SETS FOR DETECTING MAMMALIAN MICRORNA TARGETS

		Extension		Reverse
Assay Number	Target microRNA	Primer Name	Extension Primer Sequence	Primer Name	Reverse Primer Sequence	Comments

1	hmr-miR-495	Hmr-miR-	CATGATCAGCTGGGCCAAGAAAGAAGTGCA	Hmr-miR-	AAA+CAAA+CA+TGGTGCAC	Conserved across all
		495GS10	SEQ ID NO: 500	495RP1	SEQ ID NO: 504	three species
		Hmr-miR-	CATGATCAGCTGGGCCAAGAAAGAAGTGC	Hmr-miR-	AAA+C+AAA+CATGGTGC
		495GS9	SEQ ID NO: 501	495RP2	SEQ ID NO: 505
		Hmr-miR-	CATGATCAGCTGGGCCAAGAAAGAAGTG
		495GS8	SEQ ID NO: 502
		Hmr-miR-	CATGATCAGCTGGGCCAAGAAAGAAGT
		495GS7	SEQ ID NO: 503

2	mr-miR-291a-	mr-mIR-	CATGATCAGCTGGGCCAAGAGGCACACAAA	mr-mIR-291a-	AA+AG+TGCTTCCACTTTGT	Mouse/rat specific; seed
	3p	291a-	SEQ ID NO: 506	3pRP1	SEQ ID NO: 510	region ortholog to human
		3pGS10				miR-371/2
		mr-mIR-	CATGATCAGCTGGGCCAAGAGGCACACAA	mr-mIR-291a-	AA+AG+TG+CTTCCACTTT
		291a-3pGS9	SEQ ID NO: 507	3pRP2	SEQ ID NO: 511
		mr-mIR-	CATGATCAGCTGGGCCAAGAGGCACACA
		291a-3pGS8	SEQ ID NO: 508
		mr-mIR-	CATGATCAGCTGGGCCAAGAGGCACAC
		291a-3pGS7	SEQ ID NO: 509

3	m-miR-291b-	m-mIR-	CATGATCAGCTGGGCCAAGAGACAAACAAA	m-mIR-291b-	AA+AG+TG+CAT+CCATTTTGT	Mouse specific; seed
	3p	291b-	SEQ ID NO: 512	3pRP1	SEQ ID NO: 516	region ortholog to human
		3pGS10				miR-371/2
		m-mIR-	CATGATCAGCTGGGCCAAGAGACAAACAA	m-mIR-291b-	AA+AG+TG+CATCCATTTT
		291b-3pGS9	SEQ ID NO: 513	3pRP2	SEQ ID NO: 517
		m-mIR-	CATGATCAGCTGGGCCAAGAGACAAACA
		291b-3pGS8	SEQ ID NO: 514
		m-mIR-	CATGATCAGCTGGGCCAAGAGACAAAC
		291b-3pGS7	SEQ ID NO: 515

4	h-miR-519a	h-miR-	CATGATCAGCTGGGCCAAGAGTAACACTCT	h-miR-	AA+AG+TG+CATCCTTTTAGAGT	Human specific;
		519aGS10	SEQ ID NO: 518	519aRP1	SEQ ID NO: 522	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAGTAACACTC	h-miR-	AA+AG+TG+CATCCTTTTAGA
		519aGS9	SEQ ID NO: 519	519aRP2	SEQ ID NO: 523
		h-miR-	CATGATCAGCTGGGCCAAGAGTAACACT
		519aGS8	SEQ ID NO: 520
		h-miR-	CATGATCAGCTGGGCCAAGAGTAACAC
		519aGS7	SEQ ID NO: 521

5	h-miR-519b	h-miR-	CATGATCAGCTGGGCCAAGAAAACCTCTAA	h-miR-	AA+AG+TG+CATCCTTTTAG	Human specific;
		519bGS10	SEQ ID NO: 524	519bRP1	SEQ ID NO: 528	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAAAACCTCTA	h-miR-	AA+AG+TG+CATCCTTTT
		519bGS9	SEQ ID NO: 525	519bRP2	SEQ ID NO: 529
		h-miR-	CATGATCAGCTGGGCCAAGAAAACCTCT
		519bGS8	SEQ ID NO: 526
		h-miR-	CATGATCAGCTGGGCCAAGAAAACCTC
		519bGS7	SEQ ID NO: 527

6	h-miR-519c	h-miR-	CATGATCAGCTGGGCCAAGAATCCTCTAAA	h-miR-	AA+AG+TG+CATCTTTTTAGA	Human specific;
		519cGS10	SEQ ID NO: 530	519cRP1	SEQ ID NO: 534	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAATCCTCTAA	h-miR-	AA+AG+TG+CATCTTTTTA
		519cGS9	SEQ ID NO: 531	519cRP2	SEQ ID NO: 535
		h-miR-	CATGATCAGCTGGGCCAAGAATCCTCTA
		519cGS8	SEQ ID NO: 532
		h-miR-	CATGATCAGCTGGGCCAAGAATCCTCT
		519cGS7	SEQ ID NO: 533

7	h-miR-519d	h-miR-	CATGATCAGCTGGGCCAAGAACACTCTAAA	h-miR-	C+AAAG+TGCCTCCCTTTAG	Human specific;
		519dGS10	SEQ ID NO: 536	519dRP1	SEQ ID NO: 540	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAACACTCTAA	h-miR-	C+AA+AG+TGCCTCCCTTT
		519dGS9	SEQ ID NO: 537	519dRP2	SEQ ID NO: 541
		h-miR-	CATGATCAGCTGGGCCAAGAACACTCTA
		519dGS8	SEQ ID NO: 538
		h-miR-	CATGATCAGCTGGGCCAAGAACACTCT
		519dGS7	SEQ ID NO: 539

8	h-miR-520a	h-miR-	CATGATCAGCTGGGCCAAGAACAGTCCAAA	h-miR-	AA+AG+TGCTTCCCTTTGG	Human specific;
		520aGS10	SEQ ID NO: 542	520aRP1	SEQ ID NO: 546	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAACAGTCCAA	h-miR-	AA+AG+T+GCTTCCCTTT
		520aGS9	SEQ ID NO: 543	520aRP2	SEQ ID NO: 547
		h-miR-	CATGATCAGCTGGGCCAAGAACAGTCCA
		520aGS8	SEQ ID NO: 544
		h-miR-	CATGATCAGCTGGGCCAAGAACAGTCC
		520aGS7	SEQ ID NO: 545

9	h-miR-520b	h-miR-	CATGATCAGCTGGGCCAAGACCCTCTAAAA	h-miR-	AA+AG+T+GCTTCCTTTTAG	Human specific;
		520bGS10	SEQ ID NO: 548	520bRP1	SEQ ID NO: 552	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGACCCTCTAAA	h-miR-	AA+AG+TG+CTTCCTTTTA
		520bGS9	SEQ ID NO: 549	520bRP2	SEQ ID NO: 553
		h-miR-	CATGATCAGCTGGGCCAAGACCCTCTAA
		520bGS8	SEQ ID NO: 550
		h-miR-	CATGATCAGCTGGGCCAAGACCCTCTA
		520bGS7	SEQ ID NO: 551

10	h-miR-520d	h-miR-	CATGATCAGCTGGGCCAAGAAACCCACCAA	h-miR-	AA+AG+TGCTTCTCTTTGGT	Human specific;
		520dGS10	SEQ ID NO: 554	520dRP1	SEQ ID NO: 558	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAAACCCACCA	h-miR-	AA+AG+TG+CTTCTCTTTG
		520dGS9	SEQ ID NO: 555	520dRP2	SEQ ID NO: 559
		h-miR-	CATGATCAGCTGGGCCAAGAAACCCACC
		520dGS8	SEQ ID NO: 556
		h-miR-	CATGATCAGCTGGGCCAAGAAACCCAC
		520dGS7	SEQ ID NO: 557

11	h-miR-520e	h-miR-	CATGATCAGCTGGGCCAAGACCCTCAAAAA	h-miR-	AA+AG+TGCTTCCTTTTTG	Human specific;
		520eGS10	SEQ ID NO: 560	520eRP1	SEQ ID NO: 564	implicated in
						oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGACCCTCAAAA	h-miR-	AA+AG+T+GCTTCCTTTTT
		520eGS9	SEQ ID NO: 561	520eRP2	SEQ ID NO: 565
		h-miR-	CATGATCAGCTGGGCCAAGACCCTCAAA
		520eGS8	SEQ ID NO: 562
		h-miR-	CATGATCAGCTGGGCCAAGACCCTCAA
		520eGS7	SEQ ID NO: 563

12	h-miR-520f	h-miR-	CATGATCAGCTGGGCCAAGAAACCCTCTAA	h-miR-	A+AG+TGCTTCCTTTTAGA	Human specific;
		520fGS10	SEQ ID NO: 566	520fRP1	SEQ ID NO: 570	implicated in oncogenesis
		h-miR-	CATGATCAGCTGGGCCAAGAAACCCTCTA	h-miR-	A+AG+T+GCTTCCTTTTA
		520fGS9	SEQ ID NO: 567	520fRP2	SEQ ID NO: 571
		h-miR-	CATGATCAGCTGGGCCAAGAAACCCTCT
		520fGS8	SEQ ID NO: 568
		h-miR-	CATGATCAGCTGGGCCAAGAAACCCTC
		520fGS7	SEQ ID NO: 569

13	mr-miR-329	mr-miR-	CATGATCAGCTGGGCCAAGAAAAAAGGTTA	mr-miR-	AA+CA+CACCCAGCTAACC	Specific for mouse/rat
		329G510	SEQ ID NO: 572	329RP1	SEQ ID NO: 576	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGAAAAAAGGTT	mr-miR-	AA+CA+CACCCAGCTAA
		329GS9	SEQ ID NO: 573	329RP2	SEQ ID NO: 577
		mr-miR-	CATGATCAGCTGGGCCAAGAAAAAAGGT
		329GS8	SEQ ID NO: 574
		mr-miR-	CATGATCAGCTGGGCCAAGAAAAAAGG
		329GS7	SEQ ID NO: 575

14	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGAAACCCACCGA	hmr-miR-	AA+CATT+CATTGTTGTCGGT	Conserved across all
	181d	181dGS10	SEQ ID NO: 578	181dRP1	SEQ ID NO: 582	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAACCCACCG	hmr-miR-	AA+CA+TT+CATTGTTGTCG
		181dGS9	SEQ ID NO: 579	181dRP2	SEQ ID NO: 583
		hmr-miR-	CATGATCAGCTGGGCCAAGAAACCCACC
		181dGS8	SEQ ID NO: 580
		hmr-miR-	CATGATCAGCTGGGCCAAGAAACCCAC
		181dGS7	SEQ ID NO: 581

15	has-miR-193b	hmr-miR-	CATGATCAGCTGGGCCAAGAAAAGCGGGAC	hmr-miR-	AA+CT+GGCCCTCAAAGTCCC	Conserved across all
		193bGS10	SEQ ID NO: 584	193bRP1	SEQ ID NO: 588	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAAGCGGGA	hmr-miR-	AA+CT+GGCCCTCAAAGTC
		193bGS9	SEQ ID NO: 585	193bRP2	SEQ ID NO: 589
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAAGCGGG
		193bGS8	SEQ ID NO: 586
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAAGCGG
		193bGS7	SEQ ID NO: 587

16	h-miR-362	h-miR-	CATGATCAGCTGGGCCAAGAACTCACACCT	h-miR-362RP1	AAT+CCTT+GGAACCTAGGTG	Assay specific for human
		362GS10	SEQ ID NO: 590		SEQ ID NO: 594	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGAACTCACACC	h-miR-362RP2	AA+TC+CTT+GGAACCTAGG
		362GS9	SEQ ID NO: 591		SEQ ID NO: 595
		h-miR-	CATGATCAGCTGGGCCAAGAACTCACAC
		362GS8	SEQ ID NO: 592
		h-miR-	CATGATCAGCTGGGCCAAGAACTCACA
		362GS7	SEQ ID NO: 593

17	mr-miR-362	mr-mIR-	CATGATCAGCTGGGCCAAGATTCACACCTA	mr-mIR-362-	AA+TCCTT+GGAACCTAGGT	Assay specific for rodent
		362-3pGS10	SEQ ID NO: 596	3pRP1	SEQ ID NO: 600	ortholog
		mr-mIR-	CATGATCAGCTGGGCCAAGATTCACACCT	mr-mIR-362-	AA+TC+CTT+GGAACCTAG
		362-3pGS9	SEQ ID NO: 597	3pRP2	SEQ ID NO: 601
		mr-mIR-	CATGATCAGCTGGGCCAAGATTCACACC
		362-3pGS8	SEQ ID NO: 598
		mr-mIR-	CATGATCAGCTGGGCCAAGATTCACAC
		362-3pGS7	SEQ ID NO: 599

18	h-miR-500	h-miR-	CATGATCAGCTGGGCCAAGACAGAATCCTT	h-miR-500RP1	A+TG+CACCTGGGCAAGGA	Assay specific for human
		500GS10	SEQ ID NO: 602		SEQ ID NO: 606	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGACAGAATCCT	h-miR-500RP2	A+TG+CACCTGGGCAAG
		500GS9	SEQ ID NO: 603		SEQ ID NO: 607
		h-miR-	CATGATCAGCTGGGCCAAGACAGAATCC
		500GS8	SEQ ID NO: 604
		h-miR-	CATGATCAGCTGGGCCAAGACAGAATC
		500GS7	SEQ ID NO: 605

19	mmu-miR-	mr-miR-	CATGATCAGCTGGGCCAAGACTGAACCCTT	mr-miR-	A+TGCA+CCTGGGCAAGGG	Assay specific for rodent
	500	500GS10	SEQ ID NO: 608	500RP1	SEQ ID NO: 612	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGACTGAACCCT	mr-miR-	A+TGCA+CCTGGGCAAG
		500GS9	SEQ ID NO: 609	500RP2	SEQ ID NO: 613
		mr-miR-	CATGATCAGCTGGGCCAAGACTGAACCC
		500GS8	SEQ ID NO: 610
		mr-miR-	CATGATCAGCTGGGCCAAGACTGAACC
		500GS7	SEQ ID NO: 611

20	h-miR-501	h-miR-	CATGATCAGCTGGGCCAAGATCTCACCCAG	h-miR-501RP1	AA+T+CCTT+TGTCCCTGGG	Assay specific for human
		501GS10	SEQ ID NO: 614		SEQ ID NO: 618	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGATCTCACCCA	h-miR-501RP2	AAT+CCTT+TGTCCCTGG
		501GS9	SEQ ID NO: 615		SEQ ID NO: 619
		h-miR-	CATGATCAGCTGGGCCAAGATCTCACCC
		501GS8	SEQ ID NO: 616
		h-miR-	CATGATCAGCTGGGCCAAGATCTCACC
		501GS7	SEQ ID NO: 617

21	mr-miR-501	mr-miR-	CATGATCAGCTGGGCCAAGATTTCACCCAG	mr-miR-	AA+T+CC+TTTGTCCCTGGG	Assay specific for rodent
		501GS10	SEQ ID NO: 620	501RP1	SEQ ID NO: 624	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGATTTCACCCA	mr-miR-	AA+T+CC+TTTGTCCCTG
		501GS9	SEQ ID NO: 621	501RP2	SEQ ID NO: 625
		mr-miR-	CATGATCAGCTGGGCCAAGATTTCACCC
		501GS8	SEQ ID NO: 622
		mr-miR-	CATGATCAGCTGGGCCAAGATTTCACC
		501GS7	SEQ ID NO: 623

22	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGAAGTGGATGAC	hmr-miR-	AAT+CG+TACAGGGTCAT	Conserved across all
	487b	487bGS10	SEQ ID NO: 626	487bRP1	SEQ ID NO: 630	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGTGGATGA	hmr-miR-	A+AT+CG+TACAGGGTC
		487bGS9	SEQ ID NO: 627	487bRP2	SEQ ID NO: 631
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGTGGATG
		487bGS8	SEQ ID NO: 628
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGTGGAT
		487bGS7	SEQ ID NO: 629

23	h-miR-489	h-miR-	CATGATCAGCTGGGCCAAGAGCTGCCGTAT	h-miR-489RP1	AG+TGA+CATCACATATACG	Assay specific for human
		489GS10	SEQ ID NO: 632		SEQ ID NO: 636	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGAGCTGCCGTA	h-miR-489RP2	A+G+TGA+CATCACATATAC
		489GS9	SEQ ID NO: 633		SEQ ID NO: 637
		h-miR-	CATGATCAGCTGGGCCAAGAGCTGCCGT
		489GS8	SEQ ID NO: 634
		h-miR-	CATGATCAGCTGGGCCAAGAGCTGCCG
		489GS7	SEQ ID NO: 635

24	m-miR-489	m-miR-	CATGATCAGCTGGGCCAAGAGCTGCCATAT	m-miR-489RP1	AATGA+CA+CCACATATATG	Assay specific for mouse
		489GS10	SEQ ID NO: 638		SEQ ID NO: 642	ortholog
		m-miR-	CATGATCAGCTGGGCCAAGAGCTGCCATA	m-miR-489RP2	AA+TGA+CA+CCACATAT
		489GS9	SEQ ID NO: 639		SEQ ID NO: 643
		m-miR-	CATGATCAGCTGGGCCAAGAGCTGCCAT
		489GS8	SEQ ID NO: 640
		m-miR-	CATGATCAGCTGGGCCAAGAGCTGCCA
		489GS7	SEQ ID NO: 641

25	r-miR-489	r-miR-	CATGATCAGCTGGGCCAAGAGCTGCCATAT	r-miR-489RP1	AA+TGA+CA+TCACATATATG	Assay specific for rat
		489GS10	SEQ ID NO: 644		SEQ ID NO: 648	ortholog
		r-miR-	CATGATCAGCTGGGCCAAGAGCTGCCATA	r-miR-489RP2	AAT+GA+CA+TCACATATAT
		489GS9	SEQ ID NO: 645		SEQ ID NO: 649
		r-miR-	CATGATCAGCTGGGCCAAGAGCTGCCAT
		489GS8	SEQ ID NO: 646
		r-miR-	CATGATCAGCTGGGCCAAGAGCTGCCA
		489GS7	SEQ ID NO: 647

26	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGATCAACGGGAG	hmr-miR-425-	AA+TGA+CACGATCACTCCC	Conserved across all
	425-5p	425-5pGS10	SEQ ID NO: 650	5pRP1	SEQ ID NO: 654	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGATCAACGGGA	hmr-miR-425-	AA+T+GA+CACGATCACTC
		425-5pGS9	SEQ ID NO: 651	5pRP2	SEQ ID NO: 655
		hmr-miR-	CATGATCAGCTGGGCCAAGATCAACGGG
		425-5pGS8	SEQ ID NO: 652
		hmr-miR-	CATGATCAGCTGGGCCAAGATCAACGG
		425-5pGS7	SEQ ID NO: 653

27	hmr-miR-652	hmr-miR-	CATGATCAGCTGGGCCAAGATGCACAACCC	hmr-miR-	AAT+GGCGCCACTAGGGTT	Conserved across all
		652GS10	SEQ ID NO: 656	652RP1	SEQ ID NO: 660	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGATGCACAACC	hmr-miR-	AAT+GG+CGCCACTAGGG
		652GS9	SEQ ID NO: 657	652RP2	SEQ ID NO: 661
		hmr-miR-	CATGATCAGCTGGGCCAAGATGCACAAC
		652GS8	SEQ ID NO: 658
		hmr-miR-	CATGATCAGCTGGGCCAAGATGCACAA
		652GS7	SEQ ID NO: 659

28	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGAGAATTCATCA	hmr-miR-485-	AGA+GGCTGGCCGTGATG	Conserved across all
	485-5p	485-5pGS10	SEQ ID NO: 662	5pRP1	SEQ ID NO: 666	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAGAATTCATC	hmr-miR-485-	AGA+GGCTGGCCGTGA
		485-5pGS9	SEQ ID NO: 663	5pRP2	SEQ ID NO: 667
		hmr-miR-	CATGATCAGCTGGGCCAAGAGAATTCAT
		485-5pGS8	SEQ ID NO: 664
		hmr-miR-	CATGATCAGCTGGGCCAAGAGAATTCA
		485-5pGS7	SEQ ID NO: 665

29	has-miR-485-	hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGAGGAGA	hmr-miR-485-	AG+TCATA+CACGGCTCTCC	Conserved across all
	3p	485-3pGS10	SEQ ID NO: 668	3pRP1	SEQ ID NO: 672	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGAGGAG	hmr-miR-485-	AG+TC+ATACACGGCTCT
		485-3pGS9	SEQ ID NO: 669	3pRP2	SEQ ID NO: 673
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGAGGA
		485-3pGS8	SEQ ID NO: 670
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGAGG
		485-3pGS7	SEQ ID NO: 671

30	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGACGAATATAAC	hmr-miR-369-	A+GA+TC+GACCGTGTTAT	Conserved across all
	369-5p	369-5pGS10	SEQ ID NO: 674	5pRP1	SEQ ID NO: 678	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACGAATATAA	hmr-miR-369-	A+GA+TCGACCGTGTT
		369-5pGS9	SEQ ID NO: 675	5pRP2	SEQ ID NO: 679
		hmr-miR-	CATGATCAGCTGGGCCAAGACGAATATA
		369-5pGS8	SEQ ID NO: 676
		hmr-miR-	CATGATCAGCTGGGCCAAGACGAATAT
		369-5pGS7	SEQ ID NO: 677

31	hmr-miR-671	hmr-miR-	CATGATCAGCTGGGCCAAGACCTCCAGCCC	hmr-miR-	A+GGAAGCCCTGGAGGGGCT	Conserved across all
		671GS10	SEQ ID NO: 680	671RP1	SEQ ID NO: 684	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACCTCCAGCC	hmr-miR-	A+GGAAGCCCTGGAGGGG
		671GS9	SEQ ID NO: 681	671RP2	SEQ ID NO: 685
		hmr-miR-	CATGATCAGCTGGGCCAAGACCTCCAGC
		671GS8	SEQ ID NO: 682
		hmr-miR-	CATGATCAGCTGGGCCAAGACCTCCAG
		671GS7	SEQ ID NO: 683

32	h-miR-449b	h-miR-	CATGATCAGCTGGGCCAAGAGCCAGCTAAC	h-miR-	A+GGC+AGTGTATTGTTAG	Assay specific for human
		449bGS10	SEQ ID NO: 686	449bRP1	SEQ ID NO: 690	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGAGCCAGCTAA	h-miR-	AG+GC+AG+TGTATTGTT
		449bGS9	SEQ ID NO: 687	449bRP2	SEQ ID NO: 691
		h-miR-	CATGATCAGCTGGGCCAAGAGCCAGCTA
		449bGS8	SEQ ID NO: 688
		h-miR-	CATGATCAGCTGGGCCAAGAGCCAGCT
		449bGS7	SEQ ID NO: 689

33	mr-miR-449b	mr-miR-	CATGATCAGCTGGGCCAAGACCAGCTAGCA	mr-miR-	A+GGC+AGTGCATTGCTA	Assay specific for rodent
		449bGS10	SEQ ID NO: 692	449bRP1	SEQ ID NO: 696	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGACCAGCTAGC	mr-miR-	A+GG+CAGTGCATTGC
		449bGS9	SEQ ID NO: 693	449bRP2	SEQ ID NO: 697
		mr-miR-	CATGATCAGCTGGGCCAAGACCAGCTAG
		449bGS8	SEQ ID NO: 694
		mr-miR-	CATGATCAGCTGGGCCAAGACCAGCTA
		449bGS7	SEQ ID NO: 695

34	m-miR-699	m-miR-	CATGATCAGCTGGGCCAAGACGAGCCAGGT	m-miR-699RP1	A+GGCAGTGCGACCTG	Mouse specific; ortholog
		699GS10	SEQ ID NO: 698		SEQ ID NO: 702	to miR-34c
		m-miR-	CATGATCAGCTGGGCCAAGACGAGCCAGG	m-miR-699RP2	A+GG+CAGTGCGACC
		699GS9	SEQ ID NO: 699		SEQ ID NO: 703
		m-miR-	CATGATCAGCTGGGCCAAGACGAGCCAG
		699GS8	SEQ ID NO: 700
		m-miR-	CATGATCAGCTGGGCCAAGACGAGCCA
		699GS7	SEQ ID NO: 701

35	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGACAAAGTTGCT	hmr-miR-409-	A+GGT+TACCCGAGCAACT	Conserved across all
	409-5p	409-5pGS10	SEQ ID NO: 704	5pRP1	SEQ ID NO: 708	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACAAAGTTGC	hmr-miR-409-	A+GG+TTACCCGAGCAA
		409-5pGS9	SEQ ID NO: 705	5pRP2	SEQ ID NO: 709
		hmr-miR-	CATGATCAGCTGGGCCAAGACAAAGTTG
		409-5pGS8	SEQ ID NO: 706
		hmr-miR-	CATGATCAGCTGGGCCAAGACAAAGTT
		409-5pGS7	SEQ ID NO: 707

36	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGAAAGGGGTTCA	hmr-miR-409-	G+AA+TGTTGCTCGGTGAAC	Conserved across all
	409-3p	409-3pGS10	SEQ ID NO: 710	3pRP1	SEQ ID NO: 714	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAGGGGTTC	hmr-miR-409-	G+AA+TGTTGCTCGGTGA
		409-3pGS9	SEQ ID NO: 711	3pRP2	SEQ ID NO: 715
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAGGGGTT
		409-3pGS8	SEQ ID NO: 712
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAGGGGT
		409-3pGS7	SEQ ID NO: 713

37	hmr-miR-491	hmr-miR-	CATGATCAGCTGGGCCAAGACCTCATGGAA	hmr-miR-	AG+TGG+GGAACCCTTCCA	Conserved across all
		491GS10	SEQ ID NO: 716	491RP1	SEQ ID NO: 720	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACCTCATGGA	hmr-miR-	AG+TG+GGGAACCCTTC
		491GS9	SEQ ID NO: 717	491RP2	SEQ ID NO: 721
		hmr-miR-	CATGATCAGCTGGGCCAAGACCTCATGG
		491GS8	SEQ ID NO: 718
		hmr-miR-	CATGATCAGCTGGGCCAAGACCTCATG
		491GS7	SEQ ID NO: 719

38	h-miR-384	h-miR-	CATGATCAGCTGGGCCAAGATATGAACAAT	h-miR-384RP1	A+TT+CCT+AGAAATTGTTC	Assay specific for human
		384GS10	SEQ ID NO: 722		SEQ ID NO: 726	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGATATGAACAA	h-miR-384RP2	A+TT+CCT+AG+AAATTGT
		384GS9	SEQ ID NO: 723		SEQ ID NO: 727
		h-miR-	CATGATCAGCTGGGCCAAGATATGAACA
		384GS8	SEQ ID NO: 724
		h-miR-	CATGATCAGCTGGGCCAAGATATGAAC
		384GS7	SEQ ID NO: 725

39	mr-miR-384	mr-miR-	CATGATCAGCTGGGCCAAGATGTGAACAAT	mr-miR-	A+TT+CCT+AGAAATTGTT	Assay specific for rodent
		384GS10	SEQ ID NO: 728	384RP1	SEQ ID NO: 732	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGATGTGAACAA	mr-miR-	A+TT+CCT+AG+AAATTGTT
		384GS9	SEQ ID NO: 729	384RP2	SEQ ID NO: 733
		mr-miR-	CATGATCAGCTGGGCCAAGATGTGAACA
		384GS8	SEQ ID NO: 730
		mr-miR-	CATGATCAGCTGGGCCAAGATGTGAAC
		384GS7	SEQ ID NO: 731

40	hmr-miR-20b	hmr-miR-	CATGATCAGCTGGGCCAAGAACCTGCACTA	hmr-miR-	C+AA+AG+TGCTCATAGTGCA	Conserved across all
		20bGS10	SEQ ID NO: 734	20bRP1	SEQ ID NO: 738	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAACCTGCACT	hmr-miR-	CAA+AG+TG+CTCATAGTG
		20bGS9	SEQ ID NO: 735	20bRP2	SEQ ID NO: 739
		hmr-miR-	CATGATCAGCTGGGCCAAGAACCTGCAC
		20bGS8	SEQ ID NO: 736
		hmr-miR-	CATGATCAGCTGGGCCAAGAACCTGCA
		20bGS7	SEQ ID NO: 737

41	hmr-miR-490	hmr-miR-	CATGATCAGCTGGGCCAAGACAGCATGGAG	hmr-miR-	C+AA+CCTGGAGGACTCCA	Conserved across all
		490GS10	SEQ ID NO: 740	490RP1	SEQ ID NO: 744	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGCATGGA	hmr-miR-	CAA+CCT+GGAGGACTC
		490GS9	SEQ ID NO: 741	490RP2	SEQ ID NO: 745
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGCATGG
		490GS8	SEQ ID NO: 742
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGCATG
		490GS7	SEQ ID NO: 743

42	hmr-miR-497	hmr-miR-	CATGATCAGCTGGGCCAAGAACAAACCACA	hmr-miR-	C+AG+CAGCACACTGTGG	Conserved across all
		497GS10	SEQ ID NO: 746	497RP1	SEQ ID NO: 750	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAACAAACCAC	hmr-miR-	C+AG+CAGCACACTGTG
		497GS9	SEQ ID NO: 747	497RP2	SEQ ID NO: 751
		hmr-miR-	CATGATCAGCTGGGCCAAGAACAAACCA
		497GS8	SEQ ID NO: 748
		hmr-miR-	CATGATCAGCTGGGCCAAGAACAAACC
		497GS7	SEQ ID NO: 749

43	h-miR-301b	h-miR-	CATGATCAGCTGGGCCAAGATGCTTTGACA	h-miR-	C+AG+TG+CAATGATATTGTCA	Assay specific for human
		301bGS10	SEQ ID NO: 752	301bRP1	SEQ ID NO: 756	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGATGCTTTGAC	h-miR-	C+AG+TG+CAATGATATTGT
		301bGS9	SEQ ID NO: 753	301bRP2	SEQ ID NO: 757
		h-miR-	CATGATCAGCTGGGCCAAGATGCTTTGA
		301bGS8	SEQ ID NO: 754
		h-miR-	CATGATCAGCTGGGCCAAGATGCTTTG
		301bGS7	SEQ ID NO: 755

44	mr-miR-301b	mr-miR-	CATGATCAGCTGGGCCAAGATGCTTTGACA	mr-miR-	C+AG+TG+CAATGGTATTGTCA	Assay specific for rodent
		301bGS10	SEQ ID NO: 758	301bRP1	SEQ ID NO: 762	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGATGCTTTGAC	mr-miR-	C+AG+TG+CAATGGTATTGT
		301bGS9	SEQ ID NO: 759	301bRP2	SEQ ID NO: 763
		mr-miR-	CATGATCAGCTGGGCCAAGATGCTTTGA
		301bGS8	SEQ ID NO: 760
		mr-miR-	CATGATCAGCTGGGCCAAGATGCTTTG
		301bGS7	SEQ ID NO: 761

45	hmr-miR-721	hmr-miR-	CATGATCAGCTGGGCCAAGATTCCCCCTTT	hmr-miR-	C+AG+TG+CAATTAAAAGGG	Conserved across all
		721GS10	SEQ ID NO: 764	721RP1	SEQ ID NO: 768	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGATTCCCCCTT	hmr-miR-	C+AG+TG+CAATTAAAAG
		721GS9	SEQ ID NO: 765	721RP2	SEQ ID NO: 769
		hmr-miR-	CATGATCAGCTGGGCCAAGATTCCCCCT
		721GS8	SEQ ID NO: 766
		hmr-miR-	CATGATCAGCTGGGCCAAGATTCCCCC
		721GS7	SEQ ID NO: 767

46	hmr-miR-532	hmr-miR-	CATGATCAGCTGGGCCAAGAACGGTCCTAC	hmr-miR-	CA+TG+CCTTGAGTGTAGG	Conserved across all
		532GS10	SEQ ID NO: 770	532RP1	SEQ ID NO: 774	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAACGGTCCTA	hmr-miR-	CA+TG+CCTTGAGTGTA
		532GS9	SEQ ID NO: 771	532RP2	SEQ ID NO: 775
		hmr-miR-	CATGATCAGCTGGGCCAAGAACGGTCCT
		532GS8	SEQ ID NO: 772
		hmr-miR-	CATGATCAGCTGGGCCAAGAACGGTCC
		532GS7	SEQ ID NO: 773

47	h-miR-488	h-miR-	CATGATCAGCTGGGCCAAGATTGAGAGTGC	h-miR-488RP1	C+CCA+GATAATGGCACT	Assay specific for human
		488GS10	SEQ ID NO: 776		SEQ ID NO: 780	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGATTGAGAGTG	h-miR-488RP2	C+CC+A+GATAATGGCA
		488GS9	SEQ ID NO: 777		SEQ ID NO: 781
		h-miR-	CATGATCAGCTGGGCCAAGATTGAGAGT
		488GS8	SEQ ID NO: 778
		h-miR-	CATGATCAGCTGGGCCAAGATTGAGAG
		488GS7	SEQ ID NO: 779

48	mr-miR-488	mr-miR-	CATGATCAGCTGGGCCAAGATTGAGAGTGC	mr-miR-	C+CCA+GATAATAGCACT	Assay specific for rodent
		488GS10	SEQ ID NO: 782	488RP1	SEQ ID NO: 786	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGATTGAGAGTG	mr-miR-	C+CC+A+GATAATAGCA
		488GS9	SEQ ID NO: 783	488RP2	SEQ ID NO: 787
		mr-miR-	CATGATCAGCTGGGCCAAGATTGAGAGT
		488GS8	SEQ ID NO: 784
		mr-miR-	CATGATCAGCTGGGCCAAGATTGAGAG
		488GS7	SEQ ID NO: 785

49	hmr-miR-539	hmr-miR-	CATGATCAGCTGGGCCAAGAACACACCAAG	hmr-miR-	GG+AG+AAATTATCCTTGGT	Conserved across all
		539GS10	SEQ ID NO: 788	539RP1	SEQ ID NO: 792	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAACACACCAA	hmr-miR-	G+GA+G+AAATTATCCTTGG
		539GS9	SEQ ID NO: 789	539RP2	SEQ ID NO: 793
		hmr-miR-	CATGATCAGCTGGGCCAAGAACACACCA
		539GS8	SEQ ID NO: 790
		hmr-miR-	CATGATCAGCTGGGCCAAGAACACACC
		539GS7	SEQ ID NO: 791

50	h-miR-505	h-miR-	CATGATCAGCTGGGCCAAGAGAGGAAACCA	h-miR-505RP1	GT+CAA+CACTTGCTGGTT	Assay specific for human
		505GS10	SEQ ID NO: 794		SEQ ID NO: 798	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGAGAGGAAACC	h-miR-505RP2	G+T+CAA+CACTTGCTGG
		505GS9	SEQ ID NO: 795		SEQ ID NO: 799
		h-miR-	CATGATCAGCTGGGCCAAGAGAGGAAAC
		505GS8	SEQ ID NO: 796
		h-miR-	CATGATCAGCTGGGCCAAGAGAGGAAA
		505GS7	SEQ ID NO: 797

51	mr-miR-505	mr-miR-	CATGATCAGCTGGGCCAAGAGGAAACCAGC	mr-miR-	CG+T+CAA+CA+CTTGCTGGT	Assay specific for rodent
		505GS10	SEQ ID NO: 800	505RP1	SEQ ID NO: 804	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGAGGAAACCAG	mr-miR-	CG+T+CAA+CA+CTTGCTG
		505GS9	SEQ ID NO: 801	505RP2	SEQ ID NO: 805
		mr-miR-	CATGATCAGCTGGGCCAAGAGGAAACCA
		505GS8	SEQ ID NO: 802
		mr-miR-	CATGATCAGCTGGGCCAAGAGGAAACC
		505GS7	SEQ ID NO: 803

52	h-miR-18b	h-miR-	CATGATCAGCTGGGCCAAGATAACTGCACT	h-miR-18bRP1	TAA+GG+TGCATCTAGTGC	Assay specific for human
		18bGS10	SEQ ID NO: 806		SEQ ID NO: 810	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGATAACTGCAC	h-miR-18bRP2	T+AA+GG+TGCATCTAGT
		18bGS9	SEQ ID NO: 807		SEQ ID NO: 811
		h-miR-	CATGATCAGCTGGGCCAAGATAACTGCA
		18bGS8	SEQ ID NO: 808
		h-miR-	CATGATCAGCTGGGCCAAGATAACTGC
		18bGS7	SEQ ID NO: 809

53	mr-miR-18b	mr-miR-	CATGATCAGCTGGGCCAAGATAACAGCACT	mr-miR-	T+AA+GG+TGCATCTAGTGC	Assay specific for rodent
		18bGS10	SEQ ID NO: 812	18bRP1	SEQ ID NO: 816	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGATAACAGCAC	mr-miR-	TAA+GG+TG+CATCTAGT
		18bGS9	SEQ ID NO: 813	18bRP2	SEQ ID NO: 817
		mr-miR-	CATGATCAGCTGGGCCAAGATAACAGCA
		18bGS8	SEQ ID NO: 814
		mr-miR-	CATGATCAGCTGGGCCAAGATAACAGC
		18bGS7	SEQ ID NO: 815

54	hmr-miR-503	hmr-miR-	CATGATCAGCTGGGCCAAGACAGTACTGTT	hmr-miR-	T+AGC+AGCGGGAACAGT	Conserved across all
		503GS10	SEQ ID NO: 818	503RP1	SEQ ID NO: 822	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGTACTGT	hmr-miR-	T+AGC+AGCGGGAACA
		503GS9	SEQ ID NO: 819	503RP2	SEQ ID NO: 823
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGTACTG
		503GS8	SEQ ID NO: 820
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGTACT
		503GS7	SEQ ID NO: 821

55	hmr-miR-455	hmr-miR-	CATGATCAGCTGGGCCAAGACGATGTAGTC	hmr-miR-	TA+TG+TGCCTTTGGACTA	Conserved across all
		455GS10	SEQ ID NO: 824	455RP1	SEQ ID NO: 828	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACGATGTAGT	hmr-miR-	TA+TG+TGCCTTTGGAC
		455GS9	SEQ ID NO: 825	455RP2	SEQ ID NO: 829
		hmr-miR-	CATGATCAGCTGGGCCAAGACGATGTAG
		455GS8	SEQ ID NO: 826
		hmr-miR-	CATGATCAGCTGGGCCAAGACGATGTA
		455GS7	SEQ ID NO: 827

56	hmr-miR-92b	hmr-miR-	CATGATCAGCTGGGCCAAGAGAGGCCGGGA	hmr-miR-	TAT+TG+CACTCGTCCCG	Conserved across all
		92bGS10	SEQ ID NO: 830	92bRP1	SEQ ID NO: 834	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAGAGGCCGGG	hmr-miR-	TAT+TG+CACTCGTCCC
		92bGS9	SEQ ID NO: 831	92bRP2	SEQ ID NO: 835
		hmr-miR-	CATGATCAGCTGGGCCAAGAGAGGCCGG
		92bGS8	SEQ ID NO: 832
		hmr-miR-	CATGATCAGCTGGGCCAAGAGAGGCCG
		92bGS7	SEQ ID NO: 833

57	h-miR-483	h-miR-	CATGATCAGCTGGGCCAAGAAGAAGACGGG	h-miR-483RP1	T+CAC+TCCTCTCCTCCCGT	Assay specific for human
		483GS10	SEQ ID NO: 836		SEQ ID NO: 840	ortholog
		h-miR-	CATGATCAGCTGGGCCAAGAAGAAGACGG	h-miR-483RP2	T+CAC+TCCTCTCCTCCC
		483GS9	SEQ ID NO: 837		SEQ ID NO: 841
		h-miR-	CATGATCAGCTGGGCCAAGAAGAAGACG
		483GS8	SEQ ID NO: 838
		h-miR-	CATGATCAGCTGGGCCAAGAAGAAGAC
		483GS7	SEQ ID NO: 839

58	mr-miR-483	mr-miR-	CATGATCAGCTGGGCCAAGAACAAGACGGG	mr-miR-	TC+ACTCCTCCCCTCCCGT	Assay specific for rodent
		483GS10	SEQ ID NO: 842	483RP1	SEQ ID NO: 846	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGAACAAGACGG	mr-miR-	TC+ACTCCTCCCCTCCC
		483GS9	SEQ ID NO: 843	483RP2	SEQ ID NO: 847
		mr-miR-	CATGATCAGCTGGGCCAAGAACAAGACG
		483GS8	SEQ ID NO: 844
		mr-miR-	CATGATCAGCTGGGCCAAGAACAAGAC
		483GS7	SEQ ID NO: 845

59	hmr-miR-484	hmr-miR-	CATGATCAGCTGGGCCAAGAATCGGGAGGG	hmr-miR-	TCA+GGCTCAGTCCCCTC	Conserved across all
		484GS10	SEQ ID NO: 848	484RP1	SEQ ID NO: 852	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAATCGGGAGG	hmr-miR-	TC+AGGCTCAGTCCCC
		484GS9	SEQ ID NO: 849	484RP2	SEQ ID NO: 853
		hmr-miR-	CATGATCAGCTGGGCCAAGAATCGGGAG
		484GS8	SEQ ID NO: 850
		hmr-miR-	CATGATCAGCTGGGCCAAGAATCGGGA
		484GS7	SEQ ID NO: 851

60	mmu-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGACAGGCTCAAA	hmr-miR-	TC+CCTGAGGAGCCCTTTGA	Rodent specific; ortholog
	351	351GS10	SEQ ID NO: 854	351RP1	SEQ ID NO: 858	to human miR-125
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGGCTCAA	hmr-miR-	TC+CCTGAGGAGCCCTTT
		351GS9	SEQ ID NO: 855	351RP2	SEQ ID NO: 859
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGGCTCA
		351GS8	SEQ ID NO: 856
		hmr-miR-	CATGATCAGCTGGGCCAAGACAGGCTC
		351GS7	SEQ ID NO: 857

61	hmr-miR-615	hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGGGAGAC	hmr-miR-	TC+CGAGCCTGGGTCTC	Conserved across all
		615GS10	SEQ ID NO: 860	615RP1	SEQ ID NO: 864	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGGGAGA	hmr-miR-	TC+CGAGCCTGGGTC
		615GS9	SEQ ID NO: 861	615RP2	SEQ ID NO: 865
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGGGAG
		615GS8	SEQ ID NO: 862
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGAGGGA
		615GS7	SEQ ID NO: 863

62	hmr-miR-486	hmr-miR-	CATGATCAGCTGGGCCAAGACTCGGGGCAG	hmr-miR-	T+CC+TGTACTGAGCTGCC	Conserved across all
		486GS10	SEQ ID NO: 866	486RP1	SEQ ID NO: 870	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACTCGGGGCA	hmr-miR-	T+CC+TGTACTGAGCTG
		486GS9	SEQ ID NO: 867	486RP2	SEQ ID NO: 871
		hmr-miR-	CATGATCAGCTGGGCCAAGACTCGGGGC
		486GS8	SEQ ID NO: 868
		hmr-miR-	CATGATCAGCTGGGCCAAGACTCGGGG
		486GS7	SEQ ID NO: 869

63	hmr-miR-494	hmr-miR-	CATGATCAGCTGGGCCAAGAAGGTTTCCCG	hmr-miR-	T+GA+AA+CATACACGGGA	Conserved across all
		494GS10	SEQ ID NO: 872	494RP1	SEQ ID NO: 876	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGGTTTCCC	hmr-miR-	T+GA+AA+CATACACGG
		494GS9	SEQ ID NO: 873	494RP2	SEQ ID NO: 877
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGGTTTCC
		494GS8	SEQ ID NO: 874
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGGTTTC
		494GS7	SEQ ID NO: 875

64	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGACTGGCACACA	hmr-miR-493-	T+GAA+GGTCTACTGTG	Conserved across all
	493-3p	493-3pGS10	SEQ ID NO: 878	3pRP1	SEQ ID NO: 882	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACTGGCACAC	hmr-miR-493-	T+GAA+GGTCTACTGT
		493-3pGS9	SEQ ID NO: 879	3pRP2	SEQ ID NO: 883
		hmr-miR-	CATGATCAGCTGGGCCAAGACTGGCACA
		493-3pGS8	SEQ ID NO: 880
		hmr-miR-	CATGATCAGCTGGGCCAAGACTGGCAC
		493-3pGS7	SEQ ID NO: 881

65	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGAAGCCTATGGA	hmr-miR-	T+GA+GAAC+TGAATTCCATA	Conserved across all
	146b	146bGS10	SEQ ID NO: 884	146bRP1	SEQ ID NO: 888	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGCCTATGG	hmr-miR-	T+GA+GAAC+TGAATTCCA
		146bGS9	SEQ ID NO: 885	146bRP2	SEQ ID NO: 889
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGCCTATG
		146bGS8	SEQ ID NO: 886
		hmr-miR-	CATGATCAGCTGGGCCAAGAAGCCTAT
		146bGS7	SEQ ID NO: 887

66	r-miR-1	r-miR-	CATGATCAGCTGGGCCAAGATACACACTTC	r-miR-1RP1	T+G+GAA+TGTAAAGAAGTG	Assay specific for rat
		1GS10	SEQ ID NO: 890		SEQ ID NO: 894	ortholog
		r-miR-1GS9	CATGATCAGCTGGGCCAAGATACACACTT	r-miR-1RP2	T+G+GAA+TGTAAAGAAG
			SEQ ID NO: 891		SEQ ID NO: 895
		r-miR-1GS8	CATGATCAGCTGGGCCAAGATACACACT
			SEQ ID NO: 892
		r-miR-1GS7	CATGATCAGCTGGGCCAAGATACACAC
			SEQ ID NO: 893

67	h-miR-675-5p	h-miR-675-	CATGATCAGCTGGGCCAAGACACTGTGGGC	h-miR-675-	T+GGTGCGGAGAGGGCCCA	Assay specific for human
		5pGS10	SEQ ID NO: 896	5pRP1	SEQ ID NO: 900	ortholog
		h-miR-675-	CATGATCAGCTGGGCCAAGACACTGTGGG	h-miR-675-	T+GGTGCGGAGAGGGC
		5pGS9	SEQ ID NO: 897	5pRP2	SEQ ID NO: 901
		h-miR-675-	CATGATCAGCTGGGCCAAGACACTGTGG
		5pGS8	SEQ ID NO: 898
		h-miR-675-	CATGATCAGCTGGGCCAAGACACTGTG
		5pGS7	SEQ ID NO: 899

68	mr-miR-675-	mr-miR-	CATGATCAGCTGGGCCAAGAACTGTGGGCC	mr-miR-675-	T+GGTGCGGAAAGGGCC	Assay specific for rodent
	5p	675-5pGS10	SEQ ID NO: 902	5pRP1	SEQ ID NO: 906	ortholog
		mr-miR-	CATGATCAGCTGGGCCAAGAACTGTGGGC	mr-miR-675-	T+GGTGCGGAAAGGG
		675-5pGS9	SEQ ID NO: 903	5pRP2	SEQ ID NO: 907
		mr-miR-	CATGATCAGCTGGGCCAAGAACTGTGGG
		675-5pGS8	SEQ ID NO: 904
		mr-miR-	CATGATCAGCTGGGCCAAGAACTGTGG
		675-5pGS7	SEQ ID NO: 905

69	hmr-miR-668	hmr-miR-	CATGATCAGCTGGGCCAAGAGTAGTGGGCC	hmr-miR-	TG+TCACTCGGCTCGGCC	Conserved across all
		668GS10	SEQ ID NO: 908	668RP1	SEQ ID NO: 912	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAGTAGTGGGC	hmr-miR-	TG+TCACTCGGCTCGG
		668GS9	SEQ ID NO: 909	668RP2	SEQ ID NO: 913
		hmr-miR-	CATGATCAGCTGGGCCAAGAGTAGTGGG
		668GS8	SEQ ID NO: 910
		hmr-miR-	CATGATCAGCTGGGCCAAGAGTAGTGG
		668GS7	SEQ ID NO: 911

70	r-miR-346	r-miR-	CATGATCAGCTGGGCCAAGAAGAGGCAGGC	r-miR-346RP1	TGTC+TGCCTGAGTGCCTG	Assay specific for rat
		346GS10	SEQ ID NO: 914		SEQ ID NO: 918	ortholog
		r-miR-	CATGATCAGCTGGGCCAAGAAGAGGCAGG	r-miR-346RP2	TGTC+TGCCTGAGTGCC
		346GS9	SEQ ID NO: 915		SEQ ID NO: 919
		r-miR-	CATGATCAGCTGGGCCAAGAAGAGGCAG
		346GS8	SEQ ID NO: 916
		r-miR-	CATGATCAGCTGGGCCAAGAAGAGGCA
		346GS7	SEQ ID NO: 917

71	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGATTCAGTTATC	hmr-miR-542-	TG+TGA+CAGATTGATAACT	Conserved across all
	542-3p	542-3pGS10	SEQ ID NO: 920	3pRP1	SEQ ID NO: 924	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGATTCAGTTAT	hmr-miR-542-	TG+T+GA+CAGATTGATAA
		542-3pGS9	SEQ ID NO: 921	3pRP2	SEQ ID NO: 925
		hmr-miR-	CATGATCAGCTGGGCCAAGATTCAGTTA
		542-3pGS8	SEQ ID NO: 922
		hmr-miR-	CATGATCAGCTGGGCCAAGATTCAGTT
		542-3pGS7	SEQ ID NO: 923

72	hmr-miR-	hmr-miR-	CATGATCAGCTGGGCCAAGACGTGACATGATG	hmr-miR-542-	CTC+GG+GGATCATCATG	Conserved across all
	542-5p	542-5pGS10	SEQ ID NO: 926	5pRP1	SEQ ID NO: 930	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGACGTGACATG	hmr-miR-542-	C+TC+GGGGATCATCAT
		542-5pGS9	SEQ ID NO: 927	5pRP2	SEQ ID NO: 931
		hmr-miR-	CATGATCAGCTGGGCCAAGACGTGACAT
		542-5pGS8	SEQ ID NO: 928
		hmr-miR-	CATGATCAGCTGGGCCAAGACGTGACA
		542-5pGS7	SEQ ID NO: 929

73	hmr-miR-499	hmr-miR-	CATGATCAGCTGGGCCAAGAAAACATCACT	hmr-miR-	T+TAA+GA+CTTGCAGTGAT	Conserved across all
		499G510	SEQ ID NO: 932	499RP1	SEQ ID NO: 936	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAACATCAC	hmr-miR-	T+TAA+GA+CTTGCAGTG
		499GS9	SEQ ID NO: 933	499RP2	SEQ ID NO: 937
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAACATCA
		499GS8	SEQ ID NO: 934
		hmr-miR-	CATGATCAGCTGGGCCAAGAAAACATC
		499GS7	SEQ ID NO: 935

74	hmr-miR-758	hmr-miR-	CATGATCAGCTGGGCCAAGAGTTAGTGGAC	hmr-miR-	TT+TG+TGACCTGGTCCAC	Conserved across all
		758GS10	SEQ ID NO: 938	758RP1	SEQ ID NO: 942	three species
		hmr-miR-	CATGATCAGCTGGGCCAAGAGTTAGTGGA	hmr-miR-	TT+TG+T+GACCTGGTCC
		758GS9	SEQ ID NO: 939	758RP2	SEQ ID NO: 943
		hmr-miR-	CATGATCAGCTGGGCCAAGAGTTAGTGG
		758GS8	SEQ ID NO: 940
		hmr-miR-	CATGATCAGCTGGGCCAAGAGTTAGTG
		758GS7	SEQ ID NO: 941

75	hmr-miR-194	miR-	CATGATCAGCTGGGCCAAGATCCACATGGA	miR-194RP1	TG+TAA+CAGCAACTCCA	Conserved across all
		194GSP10	SEQ ID NO: 944		SEQ ID NO: 948	three species
		miR-	CATGATCAGCTGGGCCAAGATCCACATGG	miR-RP2	TG+TAA+CA+GCAACTCCAT
		194GSP9	SEQ ID NO: 945		SEQ ID NO: 949
		miR-	CATGATCAGCTGGGCCAAGATCCACATG
		194GSP8	SEQ ID NO: 946
		miR-	CATGATCAGCTGGGCCAAGATCCACAT
		194GSP7	SEQ ID NO: 947

76	hmr-miR-206	mir-	CATGATCAGCTGGGCCAAGACCACACACTT	mir-206RP1	T+GGAA+TGTAAGGAAGT	Conserved across all
		206GSP10	SEQ ID NO: 950		SEQ ID NO: 954	three species
		mir-	CATGATCAGCTGGGCCAAGACCACACACT	miR-206RP2	T+G+GAA+TGTAAGGAAGTGT
		206GSP9	SEQ ID NO: 951		SEQ ID NO: 955
		mir-	CATGATCAGCTGGGCCAAGACCACACAC
		206GSP8	SEQ ID NO: 952
		mir-	CATGATCAGCTGGGCCAAGACCACACA
		206GSP7	SEQ ID NO: 953

77	hmr-miR-1	miR-1GS10	CATGATCAGCTGGGCCAAGATACATACTTC	miR-1RP1	TG+GAA+TG+TAAAGAAGTA	Conserved across all
			(SEQ ID NO: 47)		(SEQ ID NO: 959)	three species
		miR-1GS9	CATGATCAGCTGGGCCAAGATACATACTT (SEQ	miR-1RP2	T+G+GAA+TG+TAAAGAAGT
			ID NO: 956)		(SEQ ID NO: 48)
		miR-1GS8	CATGATCAGCTGGGCCAAGATACATACT (SEQ
			ID NO: 957)
		miR-1GS7	CATGATCAGCTGGGCCAAGATACATAC (SEQ ID
			NO: 958)

78	hmr-miR-9	miR-9GS10	CATGATCAGCTGGGCCAAGATCATACAGCT	miR-9RP1	T+CTTT+GGTTATCTAGCT (SEQ	Conserved across all
			(SEQ ID NO: 960)		ID NO: 964)	three species
		miR-9G59	CATGATCAGCTGGGCCAAGATCATACAGC (SEQ	miR-9RP2	TC+TTT+GGTT+ATCTAGCTGTA
			ID NO: 961)		(SEQ ID NO: 965)
		miR-9G58	CATGATCAGCTGGGCCAAGATCATACAG (SEQ
			ID NO: 962)
		miR-9G57	CATGATCAGCTGGGCCAAGATCATACA (SEQ ID
			NO: 963)

TABLE 9

			SEQ
Assay	Target		ID
Number	MicroRNA Name	RNA target sequence	NO:

1.	hmr-miR-495	AAACAAACAUGGUGCACUUCUU	966

2.	mr-miR-291a-	AAAGUGCUUCCACUUUGUGUGCC	967
	3p

3.	m-mIR-291b-3p	AAAGUGCAUCCAUUUUGUUUGUC	968

4.	h-miR-519a	AAAGUGCAUCCUUUUAGAGUGUUAC	969

5.	h-miR-519b	AAAGUGCAUCCUUUUAGAGGUUU	970

6.	h-miR-519c	AAAGUGCAUCUUUUUAGAGGAU	971

7.	h-miR-519d	CAAAGUGCCUCCCUUUAGAGUGU	972

8.	h-miR-520a	AAAGUGCUUCCCUUUGGACUGU	973

9.	h-miR-520b	AAAGUGCUUCCUUUUAGAGGG	974

10.	h-miR-520d	AAAGUGCUUCUCUUUGGUGGGUU	975

11.	h-miR-520e	AAAGUGCUUCCUUUUUGAGGG	976

12.	h-miR-520f	AAGUGCUUCCUUUUAGAGGGUU	977

13.	mr-miR-329	AACACACCCAGCUAACCUUUUU	978

14.	hmr-miR-181d	AACAUUCAUUGUUGUCGGUGGGUU	979

15.	hmr-miR-193b	AACUGGCCCUCAAAGUCCCGCUUU	980

16.	h-miR-362	AAUCCUUGGAACCUAGGUGUGAGU	981

17.	mr-mIR-362-3p	AAUCCUUGGAACCUAGGUGUGAA	982

18.	h-miR-500	AUGCACCUGGGCAAGGAUUCUG	983

19.	mr-miR-500	AUGCACCUGGGCAAGGGUUCAG	984

20.	h-miR-501	AAUCCUUUGUCCCUGGGUGAGA	985

21.	mr-miR-501	AAUCCUUUGUCCCUGGGUGAAA	986

22.	hmr-miR-487b	AAUCGUACAGGGUCAUCCACU	987

23.	h-miR-489	AGUGACAUCACAUAUACGGCAGC	988

24.	m-miR-489	AAUGACACCACAUAUAUGGCAGC	989

25.	r-miR-489	AAUGACAUCACAUAUAUGGCAGC	990

26.	hmr-miR-425-	AAUGACACGAUCACUCCCGUUGA	991
	5p

27.	hmr-miR-652	AAUGGCGCCACUAGGGUUGUGCA	992

28.	hmr-miR-485	AGAGGCUGGCCGUGAUGAAUUC	993
	-5p

29.	hmr-miR-485	AGUCAUACACGGCUCUCCUCUCU	994
	-3p

30.	hmr-miR-369	AGAUCGACCGUGUUAUAUUCG	995
	-5p

31.	hmr-miR-671	AGGAAGCCCUGGAGGGGCUGGAGG	996

32.	h-miR-449b	AGGCAGUGUAUUGUUAGCUGGC	997

33.	mr-miR-449b	AGGCAGUGCAUUGCUAGCUGG	998

34.	m-miR-699	AGGCAGUGCGACCUGGCUCG	999

35.	hmr-miR-409-	AGGUUACCCGAGCAACUUUGCA	1000
	5p

36.	hmr-miR-409-	GAAUGUUGCUCGGUGAACCCCUU	1001
	3p

37.	hmr-miR-491	AGUGGGGAACCCUUCCAUGAGG	1002

38.	h-miR-384	AUUCCUAGAAAUUGUUCAUA	1003

39.	mr-miR-384	AUUCCUAGAAAUUGUUCACA	1004

40.	hmr-miR-20b	CAAAGUGCUCAUAGUGCAGGUAG	1005

41.	hmr-miR-490	CAACCUGGAGGACUCCAUGCUG	1006

42.	hmr-miR-497	CAGCAGCACACUGUGGUUUGU	1007

43.	h-miR-301b	CAGUGCAAUGAUAUUGUCAAAGCA	1008

44.	mr-miR-301b	CAGUGCAAUGGUAUUGUCAAAGCA	1009

45.	hmr-miR-721	CAGUGCAAUUAAAAGGGGGAA	1010

46.	hmr-miR-532	CAUGCCUUGAGUGUAGGACCGU	1011

47.	h-miR-488	CCCAGAUAAUGGCACUCUCAA	1012

48.	mr-miR-488	CCCAGAUAAUAGCACUCUCAA	1013

49.	hmr-miR-539	GGAGAAAUUAUCCUUGGUGUGU	1014

50.	h-miR-505	GUCAACACUUGCUGGUUUCCUC	1015

51.	mr-miR-505	CGUCAACACUUGCUGGUUUUCU	1016

52.	h-miR-18b	UAAGGUGCAUCUAGUGCAGUUA	1017

53.	mr-miR-18b	UAAGGUGCAUCUAGUGCUGUUA	1018

54.	hmr-miR-503	UAGCAGCGGGAACAGUACUGC	1019

55.	hmr-miR-455	UAUGUGCCUUUGGACUACAUCG	1020

56.	hmr-miR-92b	UAUUGCACUCGUCCCGGCCUC	1021

57.	h-miR-483	UCACUCCUCUCCUCCCGUCUUCU	1022

58.	mr-miR-483	UCACUCCUCCCCUCCCGUCUUGU	1023

59.	hmr-miR-484	UCAGGCUCAGUCCCCUCCCGAU	1024

60.	hmr-miR-351	UCCCUGAGGAGCCCUUUGAGCCUG	1025

61.	hmr-miR-615	UCCGAGCCUGGGUCUCCCUCU	1026

62.	hmr-miR-486	UCCUGUACUGAGCUGCCCCGAG	1027

63.	hmr-miR-494	UGAAACAUACACGGGAAACCU	1028

64.	hmr-miR-493-	UGAAGGUCUACUGUGUGCCAG	1029
	3p

65.	hmr-miR-146b	UGAGAACUGAAUUCCAUAGGCU	1030

66.	r-miR-1	UGGAAUGUAAAGAAGUGUGUA	1031

67.	h-miR-675-5p	UGGUGCGGAGAGGGCCCACAGUG	1032

68.	mr-miR-675-5p	UGGUGCGGAAAGGGCCCACAGU	1033

69.	hmr-miR-668	UGUCACUCGGCUCGGCCCACUAC	1034

70.	r-miR-346	UGUCUGCCUGAGUGCCUGCCUCU	1035

71.	hmr-miR-542-	UGUGACAGAUUGAUAACUGAAA	1036
	3p

72.	hmr-miR-542-	CUCGGGGAUCAUCAUGUCACG	1037
	5p

73.	hmr-miR-499	UUAAGACUUGCAGUGAUGUUU	1038

74.	hmr-miR-758	UUUGUGACCUGGUCCACUAACC	1039

75.	hmiR-194	UGUAACAGCAACUCCAUGUGGA	1040

76.	hmiR-206	UGGAAUGUAAGGAAGUGUGUGG	1041

77.	hmiR-1	UGGAAUGUAAAGAAGUAUGUA	1042

78.	hmiR-9	UCUUUGGUUAUCUAGCUGUAUGA	1043

Assay Format:
Several candidate primer sets shown above in TABLE 8 were tested in a high-throughput assay testing format as follows:
Each test assay (e.g., assay #75, #76, #77 and #78 listed in TABLE 8) was run in 4×4 wells of a 96 well plate, with 6 assays per 96 well plate, thereby allowing for rapid determination of the optimal primer pair for each target.
For each assay, each of the 4 candidate extension (GS) primers were tested in a separate row of the 96 well plate. Each of the 2 reverse primers were tested plus (1 nM DNA) or minus template (10 mM Tris pH 7.6, 0.1 mM EDTA, 100 ng/ul yeast total RNA).
Following reverse transcription, one set of duplicate non-template control and template samples was tested against reverse primer 1 (RP1) and the other against reverse primer 2 (RP2).
Reverse Transcriptase Assay Conditions:

- 6 μl of RT master mix was added to all 96 wells
- 2 μl of 0.5 μM GS primers was added to four successive wells
- yeast RNA in TE (10 mM Tris pH 7.6, 0.1 mM EDTA) was added to all odd-numbered wells and pre-diluted DNA templates was added to even-numbered wells

Samples were mixed well and the reverse transcriptase step was carried out, followed by dilution with 80 μl TE (10 mM Tris pH 7.6, 0.1 mM EDTA).
2 μl of the reverse transcription mixture was transferred into quadruplicate wells of a 384 well PCR plate preloaded with 80 PCR mix per well containing universal primer plus the appropriate reverse primers.
The quantitative PCR reaction results were evaluated on a real-time PCR instrument compatible with 384 well plates.
Ct values for the PCR reactions were determined based on a baseline threshold of 0.01. The sensitivity (Ct value of 1 nM template) and dynamic range (Ct of no-template control minus the Ct of the 1 nM template) were determined for each primer pair in each assay. The results of exemplary assays #75, #76, #77 and #78, listed in TABLE 8, are shown in TABLE 10 below.

TABLE 10

ASSAY RESULTS USING CANDIDATE PRIMER SETS FOR
DETECTING MIR-1, MIR-9; MIR-194 AND MIR-206

					Selected
microRNA				Dynamic	for use in
target	Extension primer	Reverse primer	Sensitivity	Range	profiling

miR-9	miR-9GS10	miR-9 RP1	13	9	−
(SEQ ID NO: 1043)	(SEQ ID NO: 960)	(SEQ ID NO: 964)
	miR-9GS9	miR-9 RP1	13	4	−
	(SEQ ID NO: 961)	(SEQ ID NO: 964)
	miR-9GS8	miR-9 RP1	10	0	−
	(SEQ ID NO:962)	(SEQ ID NO: 964)
	miR-9GS7	miR-9 RP1	16	8	−
	(SEQ ID NO: 963)	(SEQ ID NO: 964)
	miR-9GS10	miR-9 RP2	13	5	−
	(SEQ ID NO: 960)	(SEQ ID NO: 965)
	miR-9GS9	miR-9 RP2	14	4	−
	(SEQ ID NO: 961)	(SEQ ID NO: 965)
	miR-9GS8	miR-9 RP2	10	0	−
	(SEQ ID NO: 962)	(SEQ ID NO: 965)
	miR-9GS7	miR-9 RP2	17	8	−
	(SEQ ID NO: 963)	(SEQ ID NO: 965)
miR-194	miR-194GS10	miR-194RP1	9	6	−
(SEQ ID NO: 1040)	(SEQ ID NO: 944)	(SEQ ID NO: 948)
	miR-194GS9	miR-194RP1	11	5	−
	(SEQ ID NO: 945)	(SEQ ID NO: 948)
	miR-194GS8	miR-194RP1	13	17	+
	(SEQ ID NO: 946)	(SEQ ID NO: 948)
	miR-194GS7	miR-194RP1	15	17	−
	(SEQ ID NO: 947)	(SEQ ID NO: 948)
	miR-194GS10	miR-194RP2	10	6	−
	(SEQ ID NO: 944)	(SEQ ID NO: 949)
	miR-194GS9	miR-194RP2	11	6	−
	(SEQ ID NO: 945)	(SEQ ID NO: 949)
	miR-194GS8	miR-194RP2	13	16	−
	(SEQ ID NO: 946)	(SEQ ID NO: 949)
	miR-194GS7	miR-194RP2	17	16	−
	(SEQ ID NO: 947)	(SEQ ID NO: 949)
miR-1	miR-1 GS10	miR-1 RP1	15	15	−
(SEQ ID NO: 1042)	(SEQ ID NO: 47)	(SEQ ID NO: 959)
	miR-1 GS9	miR-1 RP1	17	8	−
	(SEQ ID NO: 956)	(SEQ ID NO: 959)
	miR-1 GS8	miR-1 RP1	19	11	−
	(SEQ ID NO: 957)	(SEQ ID NO: 959)
	miR-1 GS7	miR-1 RP1	22	11	−
	(SEQ ID NO: 958)	(SEQ ID NO: 959)
	miR-1 GS10	miR-1 RP2	13	15	+
	(SEQ ID NO: 47)	(SEQ ID NO: 48)
	miR-1 GS9	miR-1 RP2	15	8	−
	(SEQ ID NO: 956)	(SEQ ID NO: 48)
	miR-1 GS8	miR-1 RP2	17	11	−
	(SEQ ID NO: 957)	(SEQ ID NO: 48)
	miR-1 GS7	miR-1 RP2	19	10	−
	(SEQ ID NO: 958)	(SEQ ID NO: 48)
miR-206	miR-206 GS10	miR-206RP1	15	10	−
(SEQ ID NO: 1041)	(SEQ ID NO: 950)	(SEQ ID NO: 954)
	miR-206 GS9	miR-206RP1	16	10	−
	(SEQ ID NO: 951)	(SEQ ID NO: 954)
	miR-206 GS8	miR-206RP1	17	14	−
	(SEQ ID NO: 952)	(SEQ ID NO: 954)
	miR-206 GS7	miR-206RP1	20	20	−
	(SEQ ID NO: 953)	(SEQ ID NO: 954)
	miR-206 GS10	miR-206RP2	10	8	−
	(SEQ ID NO: 950)	(SEQ ID NO: 955)
	miR-206 GS9	miR-206RP2	11	9	−
	(SEQ ID NO: 951)	(SEQ ID NO: 955)
	miR-206 GS8	miR-206RP2	11	11	−
	(SEQ ID NO: 952)	(SEQ ID NO: 955)
	miR-206 GS7	miR-206RP2	13	20	+
	(SEQ ID NO: 953)	(SEQ ID NO: 955)

Optimal primer pairs were identified based on superior sensitivity (e.g., a preferred range between 5 and 25) and dynamic range (e.g., a preferred range between 10 and 35) characteristics. As shown above in TABLE 10, an optimal primer pair was identified for miR-194: GS8 (SEQ ID NO:946) and RP1 (SEQ ID NO:948) with a sensitivity of 13 and a dynamic range of 17. An optimal primer pair was identified for miR-1: GS10 (SEQ ID NO:47) and RP2 (SEQ ID NO:48) with a sensitivity of 13 and a dynamic range of 15. An optimal primer pair was identified for miR-206: GS7 (SEQ ID NO:953) and RP2 (SEQ ID NO:955) with a sensitivity of 13 and a dynamic range of 20. As also shown in TABLE 10, the GS primers control specificity, as shown by the significant increase in dynamic range (driven by a decrease in background) in going from GS9 to GS8 (see, e.g., miR-194).
Candidate primers designed based on the principles described above, such as the additional exemplary primers listed in TABLE 8, or other candidate primers designed using the design principles described herein, may be tested using the screening methods described above. The assays may be further optimized by using HPLC purified templates to avoid problems associated with degraded templates.
It has also been determined that microRNAs that differ from each other in sequence by only 1, 2 or 3 nucleotide changes can be readily distinguished from one another through the use of the primers designed according to the design principles and methods described herein.
While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

1. A kit for detecting at least one human microRNA selected from the group consisting of miR-21, miR-22, miR-33, miR-34a, miR-34b, miR-34c, miR-122, and miR-122a, the kit comprising at least one or more oligonucleotide primers selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.

2. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73 and SEQ ID NO:74 for detecting miR-21.

3. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:449 and SEQ ID NO:450 for detecting miR-22.

4. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:111 and SEQ ID NO:112 for detecting miR-33.

5. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO: 113,and SEQ ID NO:114 for detecting miR-34a.

6. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:116 for detecting miR-34b.

7. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:117 and SEQ ID NO:118 for detecting miR-34c.

8. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:20 for detecting miR-122.

9. The kit according to claim 1, comprising at least one primer selected from the group consisting of SEQ ID NO:147 and 148 for detecting miR-122a.

10. An oligonucleotide primer for detecting a human microRNA selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.