KR101874089B1 - A kit and method for detecting RNA molecules - Google Patents

Abstract

The present invention relates to a kit and method for detecting RNA molecules, particularly miRNA, having small sizes, such as RNA molecules. For example, the present invention provides a method of amplifying a nucleic acid molecule comprising: a first hybridizing oligonucleotide consisting of an oligo dT that hybridizes with a poly A (tail) of the RNA molecule to be amplified; and a second hybridizing oligonucleotide that is added to the 5'- end of the first hybridizing oligonucleotide A short reverse-working primer consisting of a first adapter oligonucleotide that is any nucleic acid sequence that does not hybridize to the subject RNA molecule; A second hybridizing oligonucleotide capable of specifically hybridizing with a 3'-terminal portion of the single-stranded cDNA reverse transcribed from the amplification target RNA molecule except for the oligo dT portion, and a second hybridizing oligonucleotide which is added to the 5'-end of the second hybridizing oligonucleotide An extension primer consisting of a second adapter oligonucleotide consisting of any nucleic acid sequence that is not hybridized with said single strand cDNA, said extension primer being longer than said reverse primer; And a forward primer selected in the second adapter oligonucleotide.

Description

[0001] KIT AND METHOD FOR DETECTING A &

The present invention relates to kits and methods for nucleic acid detection, and more particularly, to kits and methods for detection of nucleic acids.

MicroRNAs (abbreviated as "miRNAs") are untranslated RNAs composed of about 22 ribonucleotides, which control RNA silencing at the transcriptional stage and the expression of the gene at the post-transcriptional stage (Bartel, DP, Cell , 136 (2): 215-233, 2009). It is known that the function of such miRNAs is carried out through the formation of base pairs with complementary sequences in mRNA.

Since it was first discovered in C. elegans , it has been reported that various miRNAs are reported to be used as a regulator of gene expression in animals, plants and viruses. In addition, it is known that certain diseases such as cancer can occur due to incompatibility of functions caused by mutation of miRNA gene (Mraz, M. and Pospisilova, S., Expert Rev. Hematol ., 5 (6): 579 -581, 2012; Hughes et al, Am J. Hum Genet, 89:.. 628-633, 2011; Pontual et al, Nat Genet, 43 (10):..... 1026-1030, 2011; Lu et al ., Nature 435 (9): 834-838, 2005).

Because miRNAs are known to be very small in size (~ 22 nt) and are more easily degraded than mRNAs, there is great fear of isolating or detecting them. Typical methods for detecting this include the Northern blot analysis, hybridization-based detection using a microarray, RT-PCR using a stem loop structure complementary specifically binding to a specific miRNA, and quantitative PCR accompanied by the primer (Chen et al ., Nucleic Acids Res ., 33 (20): e179, 2005) and a method of detecting and quantifying the poly A tail at the 3'-end of miRNA with polyA polymerase , And synthesizing cDNA using a poly (T) adapter as a primer followed by amplification of miRNA using a miRNA-specific forward primer and a reverse primer based on a poly (T) adapter (Shi, R. and Chiang, VL, BioTechniques , 39: 519-525, 2005).

However, since these methods use a limited complementary sequence, nonspecific amplification may be possible, and since they are usually based on real-time PCR technology, expensive real-time PCR equipment is required. Because the generated PCR products are similar in size, it has a disadvantage that multiplex analysis for detecting miRNA at the same time is limited.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a kit and method for detecting small-sized RNA molecules containing miRNAs that are more efficient and economical to solve various problems including the above-described problems. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a method of amplifying a target RNA molecule, comprising: a first hybridization oligonucleotide consisting of oligo dT that hybridizes with a poly A (tail) of the RNA molecule to be amplified; and a second hybridization oligonucleotide A short reversal primer consisting of a first adapter oligonucleotide that is any nucleic acid sequence that does not hybridize to the RNA molecule to be amplified; A second hybridizing oligonucleotide capable of specifically hybridizing with a 3'-terminal portion of the single-stranded cDNA reverse transcribed from the amplification target RNA molecule except for the oligo dT portion, and a second hybridizing oligonucleotide which is added to the 5'-end of the second hybridizing oligonucleotide An extension primer consisting of a second adapter oligonucleotide consisting of any nucleic acid sequence that is not hybridized with said single strand cDNA, said extension primer being longer than said reverse primer; And a forward primer selected in the second adapter oligonucleotide are provided.

According to another aspect of the present invention, there is provided a method for amplifying a target RNA molecule, comprising: a first hybridization oligonucleotide consisting of a nucleic acid sequence that hybridizes with a 3'-terminal portion composed of a nucleic acid of 5 to 9 nt at the 3'- A reverse reverse primer reverse primer consisting of a first adapter oligonucleotide that is added to the 5'-end of the oligonucleotide and is any nucleic acid sequence that does not hybridize with the RNA molecule to be amplified; A second hybridization oligonucleotide capable of specifically hybridizing with a portion of the single strand cDNA reverse transcribed from the amplification target RNA molecule except for a portion corresponding to the 3'-terminal portion of the amplification target RNA molecule hybridized with the first hybridization oligonucleotide And a second adapter oligonucleotide added to the 5'-end of the second hybridizing oligonucleotide and composed of any nucleic acid sequence that is not hybridized with the single-stranded cDNA, wherein the extension primer having a longer length than the reverse primer ; And a forward primer selected in the second adapter oligonucleotide.

According to another aspect of the present invention, there is provided a method for producing a poly A tailed RNA molecule, the method comprising the steps of: preparing a poly A tail-linked RNA molecule that links a poly A tail to an RNA molecule in a sample separated from a detection target using a poly A polymerase;

A first hybridized oligonucleotide consisting of an oligo dT that hybridizes a poly A tailed RNA molecule with a poly A (poly A) tail of the poly A tailed RNA molecule, and a second hybridized oligonucleotide that hybridizes at the 5'-end of the first hybridized oligonucleotide A short reverse-working primer consisting of a first adapter oligonucleotide which is added and which is any nucleic acid sequence that does not hybridize with the poly A tail-linked RNA molecule, and a reverse transcription step of reverse transcription by a reverse transcriptase; Inactivating the reverse transcriptase, and dissolving the DNA produced by the reverse transcription to prepare a reverse transcribed single stranded cDNA from the poly A tailed RNA molecule; And

A second hybridization oligonucleotide capable of specifically hybridizing with the 3'-terminal portion of the reverse transcribed single strand cDNA except for the oligo dT portion thereof, and a second hybridization oligonucleotide capable of specifically hybridizing at the 5 'end of the second hybridization oligonucleotide, -Terminal and is not hybridized with the single-stranded cDNA, wherein the adapter primer comprises an extension primer having a longer length than the reverse primer, a second adapter oligonucleotide selected from the second adapter oligonucleotide There is provided a method for detecting an RNA molecule to be amplified comprising a primer extension comprising a forward primer and a thermostable DNA polymerase, and a PCR reaction step in which the primer extension and the PCR reaction are carried out in a single step after being transferred to a container for PCR reaction.

According to another aspect of the present invention, there is provided a method for amplifying a target RNA molecule to be amplified, comprising the steps of: (a) mixing a first hybridization oligo (first oligonucleotide) comprising a nucleic acid sequence with a 3'- And a first adapter oligonucleotide which is added to the 5'-end of the first hybridizing oligonucleotide and is not hybridized with the RNA molecule to be amplified, wherein the first adapter oligonucleotide comprises a first reverse transcription reverse primer comprising a first reverse transcription- A reverse transcriptional reaction step; Inactivating the reverse transcriptase and preparing a reverse transcribed single stranded cDNA from the amplified RNA molecule by dissolving the DNA produced by the reverse transcription; And

All or a part of the reverse transcription reaction product can be specifically hybridized with a portion of the reverse transcribed single strand cDNA other than the portion corresponding to the 3'-terminal portion of the amplification target RNA molecule hybridized with the first hybridization oligonucleotide And a second adapter oligonucleotide comprising a second hybridization oligonucleotide and any nucleic acid sequence added to the 5'-end of the second hybridization oligonucleotide and not hybridizing with the single-stranded cDNA, wherein the second adapter oligonucleotide has a length A primer extension including a long extension primer, a forward primer selected in the second adapter oligonucleotide and a thermostable DNA polymerase, and a PCR that performs a primer extension reaction and a PCR reaction in a single step after being transferred to a container for PCR reaction Detection of the RNA molecule to be amplified, including a reaction step This method is provided.

According to one embodiment of the present invention, it is possible to accurately and quickly detect a specific RNA without expensive equipments, and multiplex analysis capable of simultaneously detecting a plurality of RNAs is also possible. For example, the kit and method of the present invention can be very usefully used for diagnosis and prognosis determination of various diseases including cancer. Of course, the scope of the present invention is not limited by the above-described effects.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a method for detecting miRNA using a polyA polymerase according to an embodiment of the present invention; FIG.
FIG. 2 is a schematic diagram showing a miRNA detection method using a miRNA-specific reverse-transcription-using primer according to an embodiment of the present invention.
FIGS. 3 to 10 are graphs showing the relationship between miR-532, miR-196b, miR-196b, and miR-3b by miRNA detection kit and method using polyA polymerase and oligo dT primer, respectively, miR-362, miR-29c, miR-106b, miR-200c, miR-127 and miR-145. In each figure, the upper part shows the change of the fluorescence value according to the number of the amplification cycles, the discontinuity is the graph showing the change of the fluorescence emission rate with the temperature, and the left table shows the kind of the sample, the fluorescence detection reference value, And the right side of the lower part is a photograph showing the result of 1.5% agarose gel electrophoresis.
FIG. 11 to FIG. 18 are graphs showing the effect of the miRNA-532, miR-196b, miR-362, miR-29c, miR-106b, miR-200c, miR-127 and miR-145. In each figure, the upper left represents the change of the fluorescence value according to the number of amplification cycles, the left stop represents the change of the fluorescence emission rate with temperature, the lower left table shows the type of the sample, the fluorescence detection reference value, And the right side is a photograph showing the result of 1.5% agarose gel electrophoresis.
19 to 26 are graphs showing the effect of the miR-532, miR-362, miR-29c, miR-106b, miR-200c, miR-127 and miR-145 And shows the result of amplification. In each figure, the upper part shows the change of the fluorescence value according to the number of the amplification cycles, the discontinuity is the graph showing the change of the fluorescence emission rate with the temperature, and the left table shows the kind of the sample, the fluorescence detection reference value, And the right side of the lower part is a photograph showing the result of 1.5% agarose gel electrophoresis.
27 is a graph showing real-time PCR amplification results for miRNA let-7e using an miRNA detection kit according to an embodiment of the present invention and a commercially available miRNA kit (manufactured by Qiagen).
28 is a diagram showing a sequencing result of a real-time PCR amplification result for miRNA has-let-7e using an miRNA detection kit according to an embodiment of the present invention and a commercially available miRNA kit (manufactured by Qiagen).
29 is a graph showing a correlation between the number of copies of a template and a detection limit (Cq) cycle as a result of real-time PCR using miRNAs sequentially diluted with an miRNA detection kit according to an embodiment of the present invention. Graph.
FIG. 30 is a graph showing the results of quantitative measurement of the expression levels of miR-200a, miR-200b, and miR-200c, three subtypes of miR-200 in CAL27 cells and HSC3 cells using an miRNA detection kit according to an embodiment of the present invention FIG.
FIG. 31 is a graph showing real-time PCR results according to whether the reverse primer used in the miRNA detection kit according to an embodiment of the present invention is added during primer extension and real-time PCR, which is a second step reaction.

Definition of Terms:

The following terms are used in this document:

As used herein, the term "nucleotide" refers to a molecule of a nucleotide, which is composed of a base, a pentose, and a phosphate. The base includes adenine, guanine, cytosine, , And thymine. In the case of RNA, uracil is used instead of thymine. The pentose sugar is 2'-deoxyribose for DNA and ribose for RNA.

As used herein, the term "oligonucleotide" refers to a polymer of the nucleotide unit molecule, usually a short single-stranded nucleic acid chain consisting of up to 13 to 25 nucleotides, may refer to a nucleic acid chain consisting of less than 13 nucleotides or consisting of more than 25 nucleotides, such as -mer, 8-mer, 9-mer, 10-mer, 11-mer and 12-mer.

The term "polynucleotide " as used herein refers to a polymer chain of nucleotide units longer than the oligonucleotides, but may also be used in combination with the oligonucleotides. Polynucleotides include both single-stranded or double-stranded nucleic acid chains.

As used herein, the term "RNA molecule" is a macromolecular molecule that plays various roles in the encoding, decryption, regulation and expression of a gene. It forms a single strand and includes ribose Is distinguished from DNA which is usually composed of double strands in that a hydroxyl group is attached to the 2'-carbon of the DNA, and uracil is contained instead of thymine in the base. RNA molecules include mRNA encoding a protein, rRNA as a constituent of a ribosome, tRNA involved in translation of a protein, splicing speckle of a nucleus of a nucleus, and small RNAs found in Cajal bodies A small nucleolar RNA (snRNA) that plays a role of processing all constitutive messenger RNA (hnRNA), rRNA, tRNA, snoRNA (small nucleolar RNA) that plays a role of guiding the modification of other RNA such as the snRNA, SiRNA (small interfering RNA), an artificial RNA molecule synthesized to inhibit the expression of a specific gene, exRNA (extracellular RNA or exosomal RNA) existing in the exosome secreted out of the cell, retrotransposon and reproductive sequence PiRNAs that interact with piwi proteins associated with epigenetic and post-transcriptional gene silencing of other genetic elements in the cell to form RNA-protein complexes, and specific (Or miRNA), a small RNA molecule composed of about 22 nt, which functions to regulate gene expression.

As used herein, the term " RNA molecule to be amplified "means an RNA molecule to be detected to be amplified by PCR among various RNA molecules present in the sample obtained from the detection subject. Such amplification target RNA molecules can be used as markers for determining the presence or absence of a specific disease, susceptibility to a specific therapeutic agent, or tolerance by amplification and detection thereof.

As used herein, the term "sense strand " means a single-stranded nucleic acid molecule in the double-stranded DNA molecule that is oriented in the same direction as the direction of the code of the gene, and" antisense strand "means another single strand It is also possible to define a nucleic acid molecule as a "sense strand" and define its complementary strand as an "antisense strand", regardless of the direction of the DNA coding sequence.

The term " PCR (polymerase chain reaction) "or" nucleic acid amplification reaction "as used herein means a reaction in which a specific target nucleic acid molecule is amplified using a thermostable DNA polymerase. PCR includes, in addition to a DNA polymerase, a reaction buffer (a forward primer, a reverse primer), a dNTP mixture, and a divalent ion such as Mg ²⁺ , which are oligonucleotides capable of specifically hybridizing to a target nucleic acid Etc. are used. The DNA molecules generated by the PCR reaction are referred to herein as "amplification products. &Quot;

As used herein, the term "primer extension" means that a DNA polymerase extends a primer complementarily hybridized to a template nucleic acid of a limited length to form a non-chain reaction terminated at the 5'-end of the template nucleic acid . The DNA molecules generated by the primer extension reaction are referred to herein as "extended products ".

The term "primer" as used herein refers to an oligonucleotide or polynucleotide that is complementarily hybridized to a template nucleic acid molecule, which is used for the initiation of a PCR reaction or a primer extension reaction. A primer for a PCR reaction is a pair of a reverse primer (or an antisense primer) selected from a forward primer (or sense primer) selected from the same sense strand as the gene code advance direction of the nucleic acid molecule to be amplified and an antisense strand complementary to the sense strand And in the case of a primer extension reaction, a single extension primer is usually used.

As used herein, the term "adapter" refers to an oligonucleotide that is added to the 5'-end of a sense DNA corresponding to RNA to specifically identify a particular RNA, or a 5 ' - < / RTI > In this case, these adapters can be used as a primer sequence used for amplifying extended cDNA, and in the latter case can be used for the purpose of correcting the low Tm value which appears when a primer is constituted only by a nucleic acid sequence hybridizable with RNA. The adapter may be designed to minimize the nonspecific amplification reaction because there is no homology with the template nucleic acid to be amplified and other primers except the extension oligonucleotide.

As used herein, the term "hybridization oligonucleotide" refers to a nucleic acid molecule capable of hybridization by complementary binding with an RNA molecule to be amplified or a cDNA reverse transcribed from the amplification target RNA molecule.

As used herein, the term "universal primer" is a primer that can be amplified regardless of the type of target nucleic acid molecule to be amplified, and can be prepared using a nucleic acid sequence of a adapter oligonucleotide added by reverse transcription and primer extension reaction. In this case, a single primer set can be utilized to reduce the manufacturing cost of the kit.

As used herein, the term "forward primer" refers to a primer in which the orientation (5 '- > 3') of the corresponding primer matches the orientation of the gene. In contrast, "reverse primer" means a primer in which the direction of the corresponding primer is opposite to the direction of the gene.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided a method of amplifying a target RNA molecule comprising: a first hybridizing oligonucleotide consisting of oligo dT that hybridizes with a poly A (tail) of the RNA molecule to be amplified in a sample; and a second hybridizing oligonucleotide A short reverse-working primer consisting of a first adapter oligonucleotide that is any nucleic acid sequence that does not hybridize with the RNA molecule to be amplified; A second hybridizing oligonucleotide capable of specifically hybridizing with a 3'-terminal portion of the single-stranded cDNA reverse transcribed from the amplification target RNA molecule except for the oligo dT portion, and a second hybridizing oligonucleotide which is added to the 5'-end of the second hybridizing oligonucleotide An extension primer consisting of a second adapter oligonucleotide consisting of any nucleic acid sequence that is not hybridized with said single strand cDNA, said extension primer being longer than said reverse primer; And a forward primer selected in the second adapter oligonucleotide are provided.

In the kit, the RNA molecule to be amplified may be a small RNA molecule having a length of 18 to 180 nt, 20 to 90 nt, 20 to 70 nt, 20 to 50 nt, or 20 to 30 nt, snRNA, siRNA, exRNA, piRNA, scaRNA or miRNA.

In the kit, the sample may be epithelial, hair, biopsy, autopsy, blood, plasma, serum, saliva, urine, feces, sweat, sputum, runny nose, or tears.

The kit may further comprise a reverse primer consisting of a nucleic acid sequence selected from within the first adapter oligonucleotide, wherein the reverse primer is a universal RNA sequence comprising the same nucleic acid sequence, regardless of the type of RNA molecule to be amplified It may be a reverse primer. However, in a preferred embodiment, the additional reverse primer is not included. In this case, the reverse primer used in the reverse transcription reaction also functions as a reverse primer in the PCR reaction. Even if the reverse primer is not added in the PCR reaction, The reverse primer primer contained in the primer can be used as it is for the PCR reaction.

In the kit, the second adapter oligonucleotide may be an amplification target RNA tagging oligonucleotide according to the type of the RNA molecule to be amplified, or a common nucleic acid sequence independent of the amplification target RNA marker oligonucleotide and the amplification target RNA species. The forward primer may be a universal forward primer selected from the common oligonucleotides.

In the kit, the short reversal primer can have a length of about 29 to 34 nt, and the first adapter oligonucleotide can have a length of about 17 to 22 nt.

In the kit, the second hybridization oligonucleotide may be composed of 12 to 22 nt, 13 to 22 nt, or 14 to 22 nt, and is not hybridized with the reverse primer.

In the kit, the second adapter oligonucleotide may have a different length depending on the type of RNA to be amplified. In this case, it is possible to multiplexly detect two or more RNAs to be amplified by one reaction.

According to another aspect of the present invention, there is provided a method for amplifying a target RNA molecule, comprising: a first hybridization oligonucleotide consisting of a nucleic acid sequence that hybridizes with a 3'-terminal portion composed of a nucleic acid of 5 to 9 nt at the 3'- A reverse reverse primer reverse primer consisting of a first adapter oligonucleotide that is added to the 5'-end of the oligonucleotide and is any nucleic acid sequence that does not hybridize with the RNA molecule to be amplified; A second hybridization oligonucleotide capable of specifically hybridizing with a portion of the single strand cDNA reverse transcribed from the amplification target RNA molecule except for a portion corresponding to the 3'-terminal portion of the amplification target RNA molecule hybridized with the first hybridization oligonucleotide And a second adapter oligonucleotide added to the 5'-end of the second hybridizing oligonucleotide and composed of any nucleic acid sequence that is not hybridized with the single-stranded cDNA, wherein the extension primer having a longer length than the reverse primer ; And a forward primer selected in the second adapter oligonucleotide.

In the kit, the RNA molecule to be amplified may be a small RNA molecule having a length of 18 to 180 nt, 20 to 90 nt, 20 to 70 nt, 20 to 50 nt, or 20 to 30 nt, snRNA, siRNA, exRNA, piRNA, scaRNA or miRNA.

The kit may further comprise a reverse primer consisting of a nucleic acid sequence selected from within the first adapter oligonucleotide, wherein the reverse primer is a universal RNA sequence comprising the same nucleic acid sequence, regardless of the type of RNA molecule to be amplified It may be a reverse primer. However, in a preferred embodiment, the additional reverse primer is not included. In this case, the reverse primer used in the reverse transcription reaction also functions as a reverse primer in the PCR reaction. Even if the reverse primer is not added in the PCR reaction, The reverse primer primer contained in the primer can be used as it is for the PCR reaction.

In the kit, the second adapter oligonucleotide may be an amplification target RNA tagging oligonucleotide according to the type of the RNA molecule to be amplified, or a common nucleic acid sequence independent of the amplification target RNA marker oligonucleotide and the amplification target RNA species. The forward primer may be a universal forward primer selected from the common oligonucleotides.

Wherein the first adapter oligonucleotide has a length of about 20 to 22 nt, 17 to 22 < RTI ID = 0.0 > nt, nt, or 12 to 22 nt.

In the kit, the second adapter oligonucleotide may have a different length depending on the type of RNA to be amplified. In this case, it is possible to multiplexly detect two or more RNAs to be amplified by one reaction.

In the kit, the first hybridizing oligonucleotide may be composed of 6 to 8 nt, more preferably 6 to 7 nt, and most preferably 6 nt.

In the kit, the second hybridization oligonucleotide may be composed of 12 to 16 nt, 13 to 16 nt, or 14 to 16 nt, and is not hybridized with the reverse primer.

According to another aspect of the present invention, there is provided a method for producing a poly A tail-linked RNA molecule, the method comprising: preparing a poly A tail-linked RNA molecule using poly A polymerase to link a poly A tail to RNA molecules in a sample separated from a detection target;

A first hybridized oligonucleotide consisting of an oligo dT that hybridizes a poly A tailed RNA molecule with a poly A (poly A) tail of the poly A tailed RNA molecule, and a second hybridized oligonucleotide that hybridizes at the 5'-end of the first hybridized oligonucleotide A short reverse-working primer consisting of a first adapter oligonucleotide which is added and which is any nucleic acid sequence that does not hybridize with the poly A tail-linked RNA molecule, and a reverse transcription step of reverse transcription by a reverse transcriptase; Inactivating the reverse transcriptase, and dissolving the DNA produced by the reverse transcription to prepare a reverse transcribed single stranded cDNA from the poly A tailed RNA molecule; And

A second hybridization oligonucleotide capable of specifically hybridizing with the 3'-terminal portion of the reverse transcribed single strand cDNA except for the oligo dT portion thereof, and a second hybridization oligonucleotide capable of specifically hybridizing at the 5 'end of the second hybridization oligonucleotide, -Terminal and is not hybridized with the single-stranded cDNA, wherein the adapter primer comprises an extension primer having a longer length than the reverse primer, a second adapter oligonucleotide selected from the second adapter oligonucleotide There is provided a method for detecting an RNA molecule to be amplified comprising a primer extension comprising a forward primer and a thermostable DNA polymerase, and a PCR reaction step in which the primer extension and the PCR reaction are carried out in a single step after being transferred to a container for PCR reaction.

In the above detection method, the RNA molecule to be amplified may be a small RNA molecule having a length of 18 to 180 nt, 20 to 90 nt, 20 to 70 nt, 20 to 50 nt, or 20 to 30 nt, , snRNA, siRNA, exRNA, piRNA, scaRNA or miRNA.

In the detection method, the sample may be epithelial, hair, biopsy, autopsy, blood, plasma, serum, saliva, urine, feces, sweat, sputum, runny nose, or tears.

In the detection method, a reverse primer consisting of a nucleic acid sequence selected from the first adapter oligonucleotide in the PCR reaction step may further be used, and the reverse primer may be used regardless of the kind of the RNA molecule to be amplified Reverse primer consisting of the same nucleic acid sequence. However, in a preferred embodiment, the additional reverse primer is not included. In this case, the reverse primer used in the reverse transcription reaction also functions as a reverse primer in the PCR reaction. Even if the reverse primer is not added in the PCR reaction The reverse primer contained in the reverse transcription reaction can be used as it is in the PCR reaction step. The inventors of the present invention have found that when the reverse primer is additionally added in the PCR reaction step, an undesirable result such as a non-specific band appears, and more preferable results are obtained when the reverse primer is not added .

In the detection method, the second adapter oligonucleotide may be an amplification target RNA tagging oligonucleotide according to the type of the RNA molecule to be amplified, or a common nucleic acid sequence independent of the amplification target RNA marker oligonucleotide and the amplification target RNA species , And the forward primer may be a universal forward primer selected from the common oligonucleotides.

In this detection method, the short reverse primer can have a length of about 29 to 34 nt, and the first adapter oligonucleotide can have a length of about 17 to 22 nt.

In the detection method, the second hybridization oligonucleotide may be composed of 12 to 22 nt, 13 to 22 nt, or 14 to 22 nt, and is not hybridized with the reverse primer.

In the detection method, the second adapter oligonucleotide may have a different length depending on the type of RNA to be amplified. In this case, it is possible to multiplexly detect two or more RNAs to be amplified by one reaction.

According to another aspect of the present invention, there is provided a method for amplifying an RNA molecule to be amplified in a sample, comprising the steps of: amplifying a target RNA molecule in a sample with a first nucleic acid sequence comprising a nucleic acid sequence that hybridizes with a 3'- A reverse reverse primer reverse primer consisting of a first adapter oligonucleotide, a first adapter oligonucleotide, and a first adapter oligonucleotide, which is added to the 5'-end of the first hybridization oligonucleotide and which does not hybridize with the RNA molecule to be amplified, A reverse transcription reaction step using reverse transcription; Inactivating the reverse transcriptase and preparing a reverse transcribed single stranded cDNA from the amplified RNA molecule by dissolving the DNA produced by the reverse transcription; And

All or a part of the reverse transcription reaction product can be specifically hybridized with a portion of the reverse transcribed single strand cDNA other than the portion corresponding to the 3'-terminal portion of the amplification target RNA molecule hybridized with the first hybridization oligonucleotide And a second adapter oligonucleotide comprising a second hybridization oligonucleotide and any nucleic acid sequence added to the 5'-end of the second hybridization oligonucleotide and not hybridizing with the single-stranded cDNA, wherein the second adapter oligonucleotide comprises a second adapter oligonucleotide A primer extension comprising a long extension primer, a forward primer selected in the second adapter oligonucleotide and a thermostable DNA polymerase, and a primer extension reaction and a PCR reaction in a single step An amplification target RNA molecule comprising a PCR reaction step This detection method is provided.

In the above detection method, the RNA molecule to be amplified may be a small RNA molecule having a length of 18 to 180 nt, 20 to 90 nt, 20 to 70 nt, 20 to 50 nt, or 20 to 30 nt, , snRNA, siRNA, exRNA, piRNA, scaRNA or miRNA.

In the detection method, the sample may be epithelial, hair, biopsy, autopsy, blood, plasma, serum, saliva, urine, feces, sweat, sputum, runny nose, or tears.

In the detection method, a reverse primer consisting of a nucleic acid sequence selected from the first adapter oligonucleotide in the PCR reaction step may further be used, and the reverse primer may be used regardless of the kind of the RNA molecule to be amplified Reverse primer consisting of the same nucleic acid sequence. However, in a preferred embodiment, the additional reverse primer is not included. In this case, the reverse-use reverse primer used in the reverse transcription also serves as a reverse primer in the PCR reaction, and the reverse-use reverse primer is added The reverse primer reverse primer contained in the reverse transcription reaction may be used as it is in the PCR reaction step. The inventors of the present invention found that when the reverse primer reverse primer is additionally added in the PCR reaction step, an undesirable result such as a nonspecific band appears, and more preferable results are obtained when the reverse primer reverse primer is not added Respectively.

In the detection method, the second adapter oligonucleotide may be an amplification target RNA tagging oligonucleotide according to the type of the RNA molecule to be amplified, or a common nucleic acid sequence independent of the amplification target RNA marker oligonucleotide and the amplification target RNA species , And the forward primer may be a universal forward primer selected from the common oligonucleotides.

Wherein said short adapter reverse primer has a length of approximately 26 to 28 nt, 23 to 28 nt, or 18 to 28 nt, said first adapter oligonucleotide having approximately 20 to 22 nt, 17 To 22 nt, or 12 to 22 nt.

In the detection method, the second adapter oligonucleotide may have a different length depending on the type of RNA to be amplified. In this case, it is possible to multiplexly detect two or more RNAs to be amplified by one reaction.

In the above detection method, the first hybridizing oligonucleotide may preferably be composed of 6 to 8 nt, more preferably 6 to 7 nt, most preferably 6 nt have.

In the detection method, the second hybridization oligonucleotide may be composed of 12 to 16 nt, 13 to 16 nt, or 14 to 16 nt, and is not hybridized with the reverse-use reverse primer.

Unless specifically stated otherwise in the present invention, nucleic acid molecules composed of a plurality of oligonucleotides and / or polynucleotides are described in the order of 5'-end to 3'-end.

Hereinafter, a kit and method according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a method of detecting a small-sized RNA using a polyA polymerase according to an embodiment of the present invention. FIG. As shown in FIG. 1, the RNA molecule 101 exemplarily shows a very short nucleic acid molecule of about 20 to 30 nt, such as a miRNA. Since miRNA 101 does not have poly A tail unlike mRNA 101, when poly A polymerase and ATP are mixed and reacted in a sample containing miRNA 101, poly A tail 102 is added to miRNA 101 To form a poly A tailed miRNA (first reaction). Mixing the reverse adapter primer 105 composed of the first adapter oligonucleotide 104 composed of the nucleic acid sequence not hybridizing with the RNA molecule to be amplified and poly dT which is the first hybridization oligonucleotide 103 results in a poly A tail 102 The first adapter oligonucleotide 104 and the first hybridization oligonucleotide 103 are complementary to each other and the reverse transcription enzyme and the dNTP mixture are added to the reverse transcription reaction. A reverse transcript 110 of the target miRNA consisting of the antisense strand cDNA (111) is generated (second reaction). Then, inactivate the reverse transcriptase, dissolve the DNA, and terminate the reverse transcription, and prepare single stranded cDNA. To ensure sufficient template length for the PCR amplification reaction, the extension reaction is performed using an extension primer that is much longer than the reverse primer primer, but in order to prevent the degradation of reactivity, a single strand cDNA that is not a heteroduplex Longer extension primers are used to perform the extension reaction. After transferring all or a part of the reverse transcription product 110 to the second reaction container, the second hybridization oligonucleotide 122 having a sequence complementary to the antisense strand cDNA is introduced at the 3'-end thereof and the miRNA 101 and the antisense is hybridized with an extension primer 120 (extension primer longer than the reverse primer primer) having a 5'-terminal second adapter oligonucleotide 121, which is an arbitrary nucleic acid sequence that does not hybridize with the cDNA 111, When the primer extension reaction is performed with the polymerase, an extended double-stranded cDNA 140 is produced (the third reaction). At this time, if the length of the second adapter oligonucleotide 121 is regulated, labeling is possible for each target RNA, and multiple RNAs can be multiplexed simultaneously. The extended double-stranded cDNA 140 was amplified by PCR using a reverse primer 131 having the same nucleic acid sequence as the first adapter oligonucleotide and a forward primer 132 having the 5'-terminal nucleic acid sequence of the second adapter oligonucleotide as a template When the reaction is performed, the extended double-stranded cDNA 140 is exponentially amplified (the fourth reaction). The primer extension reaction (the third reaction) and the PCR reaction (the fourth reaction) are not performed sequentially but can be performed in one reaction. In this case, instead of the reverse primer 131, the reverse primer 105 used in the second reaction may be used as a reverse primer. In this case, in addition to the addition of the reverse primer 105 in the fourth reaction, 2 reverse transfer primer 105 contained in the second reaction product in the course of transferring part or all of the reaction product to the primer extension and PCR reaction container can be recycled in the fourth reaction.

FIG. 2 is a schematic diagram showing a method of short-length RNA detection using an RNA-specific primer (reverse primer) according to an embodiment of the present invention. As shown in FIG. 2, a first hybridizing oligonucleotide 134 capable of specifically complementarily binding with a portion of 5-7 nt, preferably 6 nt, of the 3'-end of RNA 101 is referred to as 3'- Specific reverse primer 136 (short reverse-in-use primer) constructed by 5'-terminating a first adapter oligonucleotide 135 composed of any nucleic acid sequence that is terminated and connected to it and does not hybridize with the target RNA, And reverse transcription is performed by the reverse transcriptase, an antisense strand cDNA (137) is generated (first step reaction). Then, inactivate the reverse transcriptase, dissolve the DNA, and terminate the reverse transcription, and prepare single stranded cDNA. To ensure sufficient template length for the PCR amplification reaction, the extension reaction is performed using an extension primer that is much longer than the reverse primer primer, but in order to prevent the degradation of reactivity, a single strand cDNA that is not a heteroduplex Longer extension primers are used to perform the extension reaction. The second hybridizing oligonucleotide 138 capable of specifically complementarily binding to the antisense strand cDNA 137 except for the above-mentioned 6 nt (i.e., about 16 nt) is made to be 3'-terminal, When the extension reaction is performed with the extension primer 143 containing the 5'-terminal second adapter oligonucleotide 142 which does not complementarily bind to both the antisense strand cDNA 137 and the RNA 101 as the oligonucleotide of the antisense strand cDNA 137 or the RNA 101 , Extending in both directions to produce an extension product comprising a portion of the RNA specific primer 136 and a complementary strand of the second adapter oligonucleotide 142 portion. At this time, the second adapter oligonucleotide 142 contains the 5'-region 141, which is a universal forward primer part commonly used in all the RNA detection kits, at the 5'-end and the 3'- (139) allows various RNAs to be detected at the same time in a multiplex analysis by varying the sequence and / or length depending on the type of RNA. When the extension reaction is completed, the PCR product 145 is generated by PCR using the forward primer 144 corresponding to the RNA-specific primer 136 and the 5'-region 141 in the same tube (second step reaction) . In the case of the forward primer 144, the 5'-terminal sequence of the second adapter oligonucleotide is selected from the 5'-terminal nucleic acid sequence of the second adapter oligonucleotide. When the 5'-terminal sequence of the second adapter oligonucleotide is designed to have the same sequence irrespective of the type of RNA , And becomes a universal forward primer. The reverse primer used in the reverse transcription reaction may be used as it is, and in this case, the reverse primer may be further added in the second step reaction, and the RNA primer 136 may be added to the first- The included reverse primer primer may be used as it is in the second step reaction. The PCR product 145 can be detected in the reaction process by real-time PCR and confirmed by agarose gel electrophoresis after the reaction is completed.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. It should be understood, however, that the present invention is not limited to the embodiments and examples described below, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. To fully inform the category of the invention to those who possess it. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Example : ≪ / RTI & In kit  Of RNA

The present inventors conducted a reverse transcription using an oligo dT primer after ligating polyA to miRNA according to an embodiment of the present invention and found that an extension including an miRNA specific sequence for a reverse transcription product containing reverse transcribed cDNA Biological extension reaction with real - time PCR primer and real - time PCR were performed in a single step to detect various miRNAs. In addition, according to one embodiment of the present invention, the present inventors have found that a miRNA-specific primer (short reverse-use primer) having a specific 6 nt nucleotide sequence at the 3'-end of a miRNA and a 6 nt (A longer extension primer than a reverse primer) was used to detect various miRNAs. The miRNA to be detected is shown in Table 1 below.

In the case of using the polyA polymerase, the poly A tail connection and the reverse transcription reaction were performed at 42 ° C. for 60 minutes, and then the reverse transcriptase was inactivated by heating at 70 ° C. for 10 minutes. The primer extension reaction and PCR were carried out at 95 ° C. For 15 minutes, and then repeated for 40 cycles at 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 70 ° C for 30 seconds. The melting curve was measured by heating for 10 seconds at 95 ° C and then increasing the temperature from 65 ° C to 95 ° C by 0.5 ° C per 5 seconds.

For miRNA-specific primers, reverse transcription was performed at 42 ° C for 60 minutes, and then the reverse transcriptase was inactivated by heating at 70 ° C for 10 minutes. Primer extension reaction and PCR were performed by dissolving DNA at 95 ° C for 15 minutes Then, 40 cycles were performed at 95 캜 for 10 seconds and at 60 캜 for 40 seconds. The melting curve was measured by heating for 10 seconds at 95 ° C and then increasing the temperature from 65 ° C to 95 ° C by 0.5 ° C per 5 seconds.

In addition, the amounts of reagents used for the reverse transcription reaction and the real-time PCR reaction are shown in Tables 2 and 3, respectively.

The PCR reaction was performed using a real-time PCR apparatus of BIO-RAD. In order to confirm whether the actual PCR reaction was properly performed, the band pattern was confirmed by 1.5% agarose gel electrophoresis.

Embodiments of the present invention Example MiRNA to be amplified Whether Poly A polymerase is used Reverse primer primer
(SEQ ID NO) For extension
primer
(SEQ ID NO) Forward primer Reverse primer One miR-532 Y One 10 18 19 2 miR-196b Y 11 3 miR-362 Y 12 4 miR-29c Y 13 5 miR-106b Y 14 6 miR-200c Y 15 7 miR-127 Y 16 8 miR-145 Y 17 9 miR-532 N 2 10 2 10 miR-196b N 3 11 3 11 miR-362 N 4 12 4 12 miR-29c N 5 13 5 13 miR-106b N 6 14 6 14 miR-200c N 7 15 7 15 miR-127 N 8 16 8 16 miR-145 N 9 17 9

Reverse transcription reagent usage sample volume Final concentration 2X Reaction Buffer 10 μl Reverse transcriptase (AddScript) 1 μl 1 dNTP mix (10 mM) 2 μl 1 mM The reverse primer primer (80 [mu] M) 1 μl 4 μM Template (total RNA of A549 cells, 1 μg / μl) 1 μl 1 μg Distilled water 5 μl Total volume 20 μl

Real time PCR reaction reagent usage sample volume Final concentration 2X SYBR Green PCR Master Mix 10 μl 1X The extension primer (200 nM) 1 μl 10 nM Forward primer (2 μM) 1 μl 100 nM Reverse transcriptant 1 μl Distilled water 7 μl Total volume 20 μl

Comparative Example :

The present inventors used the miRNA detection kit sold by Qiagen as a comparative example to compare the performance with the RNA detection kit according to one embodiment of the present invention.

Comparative Example Comparative Example MiRNA to be amplified Detection method One miR-532 Using anchor up dT adapter 2 miR-196b Using anchor up dT adapter 3 miR-362 Using anchor up dT adapter 4 miR-29c Using anchor up dT adapter 5 miR-106b Using anchor up dT adapter 6 miR-200c Using anchor up dT adapter 7 miR-127 Using anchor up dT adapter 8 miR-145 Using anchor up dT adapter

As a result of the above-mentioned analysis, as shown in FIG. 3 to FIG. 26, according to the RNA detection kit and method according to an embodiment of the present invention (FIGS. 3 to 18), Qiagen's commercial miRNA detection kit ), And that it is possible to detect miRNAs that are clearer and more accurate than those of the wild type. According to the RNA detection kit and method according to an embodiment of the present invention, since the reverse reaction time is shortened by using the short reverse primer, the entire PCR reaction rate can be increased and the RNA can be detected rapidly, Longer extension primers than used primers can be used to make templates of sufficient length for PCR reactions.

Experimental Example 1: Determination of base sequence of amplified PCR product

The present inventors amplified miRNA hsa let-7e against A549 cells using a method not using poly A polymerase and a miRNA detection kit sold by Qiagen as a comparative example, The base sequence for the PCR product was determined.

Specifically, a reverse primer consisting of the nucleic acid sequence shown in SEQ ID NO: 20, an extension primer consisting of the nucleic acid sequence shown in SEQ ID NO: 21 and a forward primer set forth in SEQ ID NO: 22 were used, Are set the same as those of the above embodiments.

As a result, non-specific amplification products were observed in the negative control group using a real-time PCR kit using the Qiagen kit as a comparative example as shown in FIG. 27, whereas in the case of the miRNA detection kit according to one embodiment of the present invention, Did not appear at all. As a result of performing sequencing on the amplified PCR product, as shown in FIG. 28, it is difficult to confirm whether the amplified product amplified with the miRNA detection kit of Qiagen can properly identify the base sequence and properly amplify the target miRNA However, in the case of the RNA detection kit according to one embodiment of the present invention, the nucleotide sequence could be accurately determined.

Experimental Example 2: Calibration curve of amplified PCR product

The present inventors then performed real-time PCR on a predetermined concentration of miRNA samples in order to confirm whether the level of miRNA contained in the sample can be accurately determined using an RNA detection kit according to an embodiment of the present invention , The detection limit cycle (Cq) was measured.

Specifically, miR-145 was used as the target miRNA, and the conditions described in the above examples were used for the reverse transcription and PCR conditions.

As a result, as shown in FIG. 29, it was confirmed that the Cq value exhibits an accurate exponential relationship from a very low concentration, and the correlation between the logarithm of the copy number of the miRNA and Cq is very high, such that the R2 value is 0.9979. Thus, from the above results, it can be confirmed that the kit and method of the present invention can be used not only for miRNA detection but also for accurate quantification.

Experimental Example 3: Subtype classification of miRNA

In the case of miRNAs, even the same miRNA, depending on the individual or the cell, shows differences in subtypes with slightly different sequences. Thus, the present inventors investigated whether the miRNA detection kit according to an embodiment of the present invention can discriminate miRNA subtypes.

Specifically, total RNA was isolated from CAL27 cells (ATCC CRL-2095) and HSC-3 cells by Trizol reagent and then subjected to complementary binding with 6'-terminal 6 nt of miR-200a, miR-200b and miR- (SEQ ID NOS: 23 to 25) composed of the 3'-terminal first hybridizing oligonucleotide and the 5'-terminal first adapter oligonucleotide were subjected to a reverse transcription reaction at 42 DEG C for 60 minutes. Then, 1 μl of the reverse-transcription reaction product was transferred to a container containing the real-time PCR reaction solution described in Table 3, and the mixture was heated at 70 ° C. for 10 minutes to inactivate the enzyme. The primer extension reaction using BioRad CFX96 Touch Real- And PCR can yield reliable results.

Experimental Example 4: Results according to addition or absence of reverse transcription primer in real-time PCR reaction

In addition, we performed primer extension and real-time PCR reactions in a single step, not separately. As a primer pair for the real-time PCR reaction, a forward primer using a unique nucleic acid sequence at the 5'-terminal of the extension primer and a reverse primer used in a reverse reaction were used as a reverse primer. However, since the reverse primer has already been used in the first step reverse transcription reaction and is contained in the first step reverse transcription reaction added to the second step primer extension and the real time PCR reaction, a reverse primer is separately added to the two step primer extension and real time PCR reaction The second-step reaction would normally be performed. The effect of two conditions on the real - time PCR reaction was investigated when the reverse primer was not added and when the reverse primer was added in the second step reaction.

Specifically, miR-16 was used as an amplification target RNA, A549 lung carcinoma cell line was used as a cell, and when no reverse primer was added, reverse transcription was performed using the conditions described in Tables 2 and 3, Primer extension and real-time PCR reaction were carried out. When the reverse primer primer was further added, 1 μl of the reverse primer primer (2 μM) was further added, and the second-step reaction was performed under the condition that the amount of distilled water was decreased by the corresponding volume . As the reverse primer, an oligonucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 29 was used. As the miR-16 specific extension primer for primer extension and real time PCR reaction, oligonucleotides composed of the nucleic acid sequence shown in SEQ ID NO: , And a forward primer consisting of a nucleic acid sequence represented by SEQ ID NO: 22 was used as a forward primer. The conditions of the reverse transcription reaction step, the primer extension and the real time PCR reaction step were the same as those of Experimental Example 2 above.

As a result, as shown in FIG. 31, when the reverse primer was added in the second reaction, nonspecific reaction products were also generated in the negative control, whereas when the reverse primer was not added, Was not detected at all. Therefore, it was found that it is more preferable to not add the reverse primer in the second step reaction.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

101: RNA molecule to be amplified
102: Poly A tail
103: First hybridization oligonucleotide (poly (dT))
104: first adapter oligonucleotide
105: Reverse primer primer
110: reverse transcriptase
111: Antisense strand cDNA
120: Extending primer
121: Second adapter oligonucleotide
122: second hybridization oligonucleotide
131: reverse primer
132: Forward primer
134: RNA-specific first hybridization oligonucleotide
135: first adapter oligonucleotide
136: Reverse primer primer
137: Reverse transcription product
138: second hybridization oligonucleotide
139: RNA-specific tagged oligonucleotide
141: universal forward primer selection site
142: Second adapter oligonucleotide
143: Extending primer
144: Universal forward primer

<110> HeimBiotek Inc. <120> A kit and method for detecting RNA molecules <130> PD16-5400 <160> 30 <170> KoPatentin 3.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer for reversetranscription with polyA tailing <400> 1 gcaccaccac tgattagttt ttttttttt 29 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-532 <400> 2 gcgagcatgc agacggtc 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-196b <400> 3 gctagtcatg ccagcccaac 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-362 <400> 4 gcgagcatcg cagactcac 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-29c <400> 5 gcgagcatcg cagtaaccg 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-106b <400> 6 gcgagcactg cacatctgc 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-200c <400> 7 gcgagcactt cacgtccatc 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-127 <400> 8 gcgacgatgc agagccaa 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for the reverse transcription and reverse primer for PCR          hsa-miR-145 <400> 9 gcgagtccgc atagagggat 20 <210> 10 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-532 <400> 10 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gcatgccttg agtgtag 77 <210> 11 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-196b <400> 11 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gtaggtagtt tcctgtt 77 <210> 12 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-362 <400> 12 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gaatccttgg aacctaggt 79 <210> 13 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-29c <400> 13 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gtagcaccat ttgaaat 77 <210> 14 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-106b <400> 14 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gtaaagtgct gacagt 76 <210> 15 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-200c <400> 15 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gtaatactgc cgggtaat 78 <210> 16 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-127 <400> 16 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 gtcggatccg tctgagc 77 <210> 17 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-miR-145 <400> 17 gcacctccag gaccaatctt gtagccagga tcttgccatc ctatggaact gcctcggtga 60 ggtccagttt tcccagga 78 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> universal forward primer for polyA tailed miRNA <400> 18 gcacctccag gaccaatc 18 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> universal reverse primer for polyA tailed miRNA <400> 19 gcaccaccac tgattag 17 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> RT primer for hsa-let-7e-5p <400> 20 tgcagggtgg cagccaacta t 21 <210> 21 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> extension primer for hsa-let-7e-5p <400> 21 taagttccgc tgtgtgccgc atctcaccgg gcggcgcttt gagcacggtg tgacggtgag 60 gtaggagtt gt 72 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> universal foward primer <400> 22 taagttccgc tgtgtgccgc 20 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RT primer for miR-200a <400> 23 tgcagggtgg cagccgctac a 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RT primer for miR-200b <400> 24 tgcagggtgg cagccactac t 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RT primer for miR-200c <400> 25 tgcagggtgg cagccctacc t 21 <210> 26 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> extension primer for miR-200a <400> 26 taagttccgc tgtgtgccgc atctcaccgg gcggcgcttt gagcacggtg tgacggtaac 60 actgtctggt aa 72 <210> 27 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> extension primer for miR-200b <400> 27 taagttccgc tgtgtgccgc atctcaccgg gcggcgcttt gagcacggtg tgacggtaat 60 actgcctggt aa 72 <210> 28 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> extension primer for miR-200c <400> 28 taagttccgc tgtgtgccgc atctcaccgg gcggcgcttt gagcacggtg tgacggtaat 60 actgccgggt aat 73 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RT primer for miR-16 <400> 29 tgcagggtgg cagcccgcca a 21 <210> 30 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> extension primer for miR-16 <400> 30 taagttccgc tgtgtgccgc atctcaccgg gcggcgcttt gagcacggtg tgacggtagc 60 agcacgtaaa ta 72

Claims (27)

delete delete delete delete delete delete delete delete delete delete delete delete (a) preparing a poly A tail-linked RNA molecule linking the poly A tail to an RNA molecule using a poly A polymerase;
(b) a first hybridizing oligonucleotide consisting of an oligo dT that hybridizes a poly A tailed RNA molecule with a poly A (tail) A of the poly A tailed RNA molecule, and a second hybridized oligonucleotide that hybridizes to the 5'end of the first hybridized oligonucleotide, - a reverse transcription primer consisting of a first adapter oligonucleotide which is a nucleic acid sequence added to the end of the poly A tailing RNA molecule and which does not hybridize with the poly A tail-linked RNA molecule, and a reverse transcription step of reverse transcription by a reverse transcriptase;
(c) inactivating the reverse transcriptase, and dissolving the DNA produced by the reverse transcription to prepare a reverse transcribed single strand cDNA from the polyA tail-ligated RNA molecule; And
(d) a second hybridization oligonucleotide capable of specifically hybridizing all or part of the reverse transcription reaction product with the 3'-terminal portion of the transcribed single strand cDNA except for the oligo dT portion, and a second hybridization oligonucleotide Of the first adapter oligonucleotide and a second adapter oligonucleotide added to the 5'-end of the second adapter oligonucleotide and consisting of any nucleic acid sequence that is not hybridized with the single strand cDNA, And a PCR reaction step in which the primer extension and the PCR reaction are carried out in a single step after transferring to a primer extension and PCR reaction container containing a forward primer and a thermostable DNA polymerase selected from SEQ ID NO:
In the step (d), no reverse primer is additionally added, and the reverse primer used in the reverse transcription reaction in the step (b) also serves as a reverse primer in the PCR reaction. Thus, Wherein the used primer is used as a reverse primer in the PCR reaction step. (a) a first hybridizing oligonucleotide consisting of a nucleic acid sequence which hybridizes an RNA molecule to be amplified with a 3'-terminal portion composed of a nucleic acid of 5 to 9 nt at the 3'-terminal of the RNA molecule to be amplified, A reverse-transcription-using reverse primer consisting of a first adapter oligonucleotide which is added to the 5'-end of the hybridization oligonucleotide and is an arbitrary nucleic acid sequence that does not hybridize with the RNA molecule to be amplified, and a reverse transcription reaction step using reverse transcriptase;
(b) inactivating the reverse transcriptase, and dissolving the DNA produced by the reverse transcription to prepare a reverse transcribed single stranded cDNA from the RNA molecule to be amplified; And
(c) amplifying all or part of the reverse-transcription reaction product with a portion excluding the portion corresponding to the 3'-terminal portion of the RNA molecule to be amplified which is hybridized with the first hybridizing oligonucleotide in the reverse transcribed single- And a second adapter oligonucleotide consisting of any nucleic acid sequence added to the 5'-end of said second hybridizing oligonucleotide and not hybridizing to said single stranded cDNA, wherein said second adapter oligonucleotide is capable of hybridizing to said first adapter oligonucleotide, Primer extension and primer extension reaction comprising a forward primer and a thermostable DNA polymerase selected in the second adapter oligonucleotide and a PCR reaction container, followed by a primer extension reaction and a PCR reaction in a single step Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
In the step (c), a reverse primer is not additionally added. The reverse primer reverse primer used in the reverse transcription reaction in the step (a) also serves as a reverse primer in the PCR reaction. Thus, Wherein the reverse primer reverse primer is used as a reverse primer in the PCR reaction step. The method according to claim 13 or 14,
Wherein the amplification target RNA molecule is a small RNA molecule having a length of 18 to 180 nt. 16. The method of claim 15,
Wherein the small RNA molecule is a tRNA, snRNA, siRNA, exRNA, piRNA, scaRNA or miRNA. The method according to claim 13 or 14,
Wherein the second adapter oligonucleotide comprises an amplification target RNA tagging oligonucleotide according to the kind of RNA molecule to be amplified or a common oligonucleotide consisting of a common nucleic acid sequence independent of the amplification target RNA marker oligonucleotide and the type of target RNA to be amplified &Lt; / RTI &gt; 14. The method of claim 13,
Wherein said reverse primer is comprised between 29 and 34 nt. 14. The method of claim 13,
Wherein the first adapter oligonucleotide is comprised between 17 and 22 nt. 15. The method of claim 14,
Wherein said reverse primer reverse primer has a length of 18-28 nt. 15. The method of claim 14,
Wherein the first adapter oligonucleotide has a length of 12-22 nt. delete delete delete delete delete delete

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