JP2006042804A - 18s ribosome rna gene originated from callithrix jacchus and its utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide "a polynucleotide as an inner standard used as a standard of a gene expression level in test samples for correcting a transfer product quantity, when a difference between the expression levels of desired genes in two or more test samples is measured as a difference between the transfer products of the genes" or the like, characterized by having a base sequence belonging to a base sequence group comprising a specific base sequence and the like, since the use of the polynucleotide is essential on a gene expression analysis. <P>SOLUTION: This 18S ribosome RNA gene originated from Callithrix Jacchus, and the 18S ribosome RNA gene originated from Callithrix Jacchus or its partial fragment are provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an 18S ribosomal RNA gene derived from a common marmoset and use thereof.

Because of species differences, it is difficult to fully evaluate the effects of drugs on humans, such as chemical substances that are active ingredients of pharmaceuticals, and the effects of toxicity and side effects in rodents such as rats and mice. The use of animals is required.
Currently, large monkeys such as rhesus monkeys and cynomolgus monkeys are used for this purpose, but small monkeys are attracting attention as experimental animals in terms of cost, operability, and the like (see, for example, Non-Patent Document 1).
On the other hand, in research using rodents such as rats and mice, it is related to gene expression analysis for the purpose of examining the effects of drugs on humans, such as chemical substances that are active ingredients of pharmaceuticals, and the effects of toxicity and side effects. Research has become active (see, for example, Non-Patent Document 2).
In the above research trends, even in small monkeys that are attracting attention as experimental animals, gene expression is aimed at more precisely examining the effects of drugs on humans, such as chemical substances that are active ingredients of pharmaceuticals, and effects such as toxicity and side effects on humans. Research on analysis will become even more important.

Mansfield, K., Comp. Med., 53, 383 (2003) Hamadeh, H. K. et al, Tox. Sci., 67, 219 (2002)

  By the way, in such gene expression analysis, when using different test samples, particularly test samples derived from different individuals or different types of tissues, transcripts of each gene contained in the test sample. Since the amount varies greatly, “when the difference in the expression level of a desired gene in two or more kinds of test samples is measured as the difference in the transcription amount of the gene, the amount of the transcription product is corrected. Use of “polynucleotide as an internal standard that serves as a reference for the expression level of the gene in the test sample”. However, in small monkeys, such a polynucleotide is not sufficiently known at the present time, and the rapid acquisition and development of the polynucleotide has been desired.

  As a result of earnest examination under such circumstances, the present inventors paid attention to a sub-small monkey called Common Marmoset (Callithrix Jacchus), which is a kind of New World monkey among small monkeys that are attracting attention as experimental animals, Indispensable in the research on gene expression analysis using the monkey, “when measuring the difference in the expression level of a desired gene in two or more kinds of test samples as the difference in the transcription level of the gene, the transcript The common marmoset-derived 18S ribosomal RNA gene was found as “polynucleotide as an internal standard that serves as a reference for the expression level of the gene in the test sample for correcting the amount”, and the present invention was achieved.

That is, the present invention
1. 18S ribosomal RNA gene derived from common marmoset (hereinafter sometimes referred to as gene of the present invention);
2. A polynucleotide comprising an 18S ribosomal RNA gene derived from a common marmoset or a partial fragment thereof, and having any of the following base sequences (hereinafter sometimes referred to as the polynucleotide of the present invention):
(1) The base sequence represented by SEQ ID NO: 1 (2) The base sequence having 95% or more base identity to the base sequence represented by SEQ ID NO: 1 (3) The description of (1) to (2) above 2. a base sequence complementary to any one of the base sequences (4) a partial base sequence in any one of the base sequences described in (1) to (3) above; A polynucleotide comprising any one of the following base sequences: (1) a base sequence represented by base numbers 1274 to 1873 of the base sequence represented by SEQ ID NO: 1 (2) a base sequence represented by SEQ ID NO: 1 Selected from the region specified by the base sequence represented by base numbers 1274 to 1873, and having any continuous base sequence having a base length of 20 to 60 (3) according to the above (1) to (6) A base sequence complementary to any base sequence;
4). DNA or a composition for detection, comprising the polynucleotide according to 2 or 3 above (hereinafter also referred to as the composition of the present invention);
5. 4. The composition according to item 4 above, wherein the polynucleotide according to item 2 or 3 is immobilized on a carrier;
6. A gene in the test sample for correcting the transcription product amount when measuring the difference in the expression level of the desired gene in two or more test samples as a difference in the transcription product amount of the gene Use of the polynucleotide according to the item 2 or 3 as an internal standard serving as a reference for the expression level of the protein (hereinafter sometimes referred to as use of the present invention);
7). Use of the polynucleotide according to item 6 above, wherein the test sample is a sample derived from common marmoset;
8. A method for measuring a difference in the expression level of a desired gene in two or more kinds of test samples as a difference in the transcription product amount of the gene,
(1) a first step of measuring the amount of transcript of the 18S ribosomal RNA gene derived from common marmoset using the polynucleotide according to item 2 or 3;
(2) a second step of measuring the amount of a transcription product of the gene using a desired gene in the test sample;
(3) In each test sample in two or more types of test samples, the amount of the transcript of 18S ribosomal RNA gene measured in the first step in any test sample is compared with the first step in other test samples. A third step of calculating the relative ratio of the transcript amount of the measured 18S ribosomal RNA gene;
(4) By multiplying the amount of transcript of the desired gene measured in the second step by the relative ratio calculated in the third step, the second step in each test sample in two or more types of test samples A fourth step of correcting the measured transcript amount of the desired gene,
A measurement method characterized by comprising (hereinafter, also referred to as the measurement method of the present invention);
9. 9. The measuring method according to item 8 above, wherein the test sample is a sample derived from a common marmoset;
10. 8. The method according to item 8 above, wherein the method for measuring the amount of a gene transcript in the first step and / or the second step is a DNA array method or a quantitative reverse transcriptase-polymerase chain reaction method. Or the measuring method according to 9;
Etc. are provided.

  According to the present invention, an 18S ribosomal RNA gene derived from a common marmoset, use thereof, and the like can be provided.

Hereinafter, the present invention will be described in detail.
The small monkey used in the present invention is a common marmoset (Callithrix Jacchus) which is a kind of New World monkey. The monkeys are ultra-compact monkeys belonging to monkeys that are closely related to humans, and have better reproductive efficiency than small monkeys, and are small and easy to handle.

  The gene of the present invention is an 18S ribosomal RNA gene derived from a common marmoset. Since the gene has a property as a housekeeping gene, the expression of the gene is hardly affected by various drug treatments, the presence or absence of a disease, physiological stimulation, etc. The product is universally and constantly expressed in living tissues. Therefore, by using the expression level of the gene as an internal standard serving as a reference for the expression level of the gene in the test sample, it becomes possible to correct the transcription product amount of the desired gene. Of course, when a gene expression analysis is performed for a specific biological species (for example, a marmoset species to which a common marmoset belongs, etc.), the transcription product of the gene using the amount of the transcription product of the housekeeping gene peculiar to the biological species. It is particularly preferred to correct the amount.

The polynucleotide of the present invention is an 18S ribosomal RNA gene derived from a common marmoset or a partial fragment thereof, and has the following base sequence:
(1) The base sequence represented by SEQ ID NO: 1 (2) The base sequence having 95% or more base identity with respect to the base sequence represented by SEQ ID NO: 1 (3) As described in (1) to (2) above A base sequence complementary to any base sequence (4) A partial base sequence in any one of the base sequences described in (1) to (3) above

  Here, the “partial base sequence” represented by item number (4) is a base sequence constituting the gene of the present invention or a primer or probe used for obtaining or detecting the gene, and item numbers (1) to (3) A base sequence having a shorter length than any one of the base sequences described in the above. Specific examples include a base sequence represented by SEQ ID NO: 2, a base sequence represented by SEQ ID NO: 3, and the like.

  In the present invention, “base identity” refers to sequence identity and homology between two DNAs or RNAs. The “identity” is determined by comparing two sequences that are optimally aligned over the entire region of the sequence to be compared. Here, the DNA or RNA to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The identity is measured using sequence analysis software, specifically analysis tools provided by Vector NTI, GENETYX-MAC, and public databases, for example, the homepage address http: // Generally available at www.ddbj.nig.ac.jp.

  “Base identity” in the present invention is based on a base sequence, and is preferably about 95% or more, for example.

The gene of the present invention or the polynucleotide of the present invention may be obtained by using a hybridization method, a PCR method or the like, but may be obtained by any method as long as it can be obtained.
For example, genetic engineering described in Sambrook, J. et al., Molecular Cloning: A laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001) or the like from marmoset tissues such as common marmoset or cultured cells derived from these animals. It can be obtained according to the method.

First, total RNA derived from marmoset tissues such as common marmoset or animal-derived cultured cells is prepared.
Specifically, for example, tissue such as liver of common marmoset is pulverized in a solution containing a protein denaturant such as guanidine hydrochloride or guanidine thiocyanate, and the protein is denatured by adding phenol, chloroform or the like to the pulverized product. After the denatured protein is removed by centrifugation, total RNA is extracted from the collected supernatant fraction by a method such as guanidine hydrochloride / phenol method, SDS-phenol method, guanidine thiocyanate / CsCl method or the like. Examples of commercially available kits based on these methods include Trizol reagent (Invitrogen), Isogen (manufactured by Nippon Gene) and the like.
Next, using the obtained total RNA as a template, an oligo dT primer is annealed to the poly A sequence of RNA, and single-stranded cDNA is synthesized by reverse transcriptase. It is also possible to synthesize double-stranded cDNA using E. coli DNA polymerase I using single-stranded cDNA as a template and RNA fragments obtained by adding nicks and gaps to the RNA strand using E. coli RNaseH. it can. The obtained cDNA is purified and recovered by usual methods such as phenol chloroform extraction and ethanol precipitation. Examples of commercially available kits based on these methods include cDNA synthesis system plus (Amersham Biosciences) and SuperScript Choice System (Invitrogen).
Next, the obtained double-stranded cDNA can be ligated using, for example, a vector such as plasmid pUC118 or phage λgt10 and ligase to prepare a cDNA library.
Also, from common marmoset tissue pieces or their animal-derived cultured cells, for example, Sambrook, J. et al., Molecular Cloning: A laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001) etc., Masamura Muramatsu, Genomic DNA is prepared according to the usual method described in "Lab Manual Genetic Engineering" (Maruzen 1998).
For example, if the sample is liver, extract buffer [10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0), 20 μg / ml, 0.5% SDS] 10 times the tissue weight Add (v / w), grind, and add Proteinase K to a final concentration of 100 μl / ml and mix. The mixture is kept at 50 ° C. for 3 hours, and then the protein is denatured by adding phenol / chloroform or the like. After the denatured protein is removed by centrifugation or the like, ethanol is added to the collected supernatant fraction to precipitate and collect genomic DNA. When the sample is peripheral blood, genomic DNA can be obtained by treating the sample with a DNA-Extraction kit (Stratagene). A genomic DNA library can be obtained by ligating the obtained genomic DNA with a vector such as λgt10 using a ligase.

From the single-stranded or double-stranded cDNA itself as described above, a cDNA library or a genomic DNA library, a partial base sequence of the base sequence represented by SEQ ID NO: 1 or a base sequence complementary to the partial base sequence PCR reaction using an oligonucleotide having as a primer, or a hybridization method using a DNA having the base sequence represented by SEQ ID NO: 1 or a partial base sequence of the base sequence as a probe from the above cDNA library or genomic DNA library, The gene of the present invention or the polynucleotide of the present invention can be obtained.
As a primer used for PCR, for example, an oligonucleotide having a length of about 15 to about 50 bases and having a base sequence starting from the 5 ′ end of the base sequence represented by SEQ ID NO: 1, An oligonucleotide having a base sequence complementary to the base sequence starting from the 3 ′ end side of the base sequence represented by SEQ ID NO: 1 can be mentioned. Specifically, examples of the forward primer include an oligonucleotide having the base sequence represented by SEQ ID NO: 2. Moreover, as a reverse primer, the oligonucleotide which consists of a base sequence shown by sequence number 3 can be mentioned, for example. As PCR conditions, for example, 5 μl of 10-fold concentrated buffer solution for Ex-Taq polymerase (manufactured by Takara Bio Inc.), 2.5 mM dNTP mixture (each 2.5 mM dATP, dGTP, dCTP and dTTP) in 50 μl of the reaction solution 5 μl (final concentrations of dATP, dGTP, dCTP and dTTP are each 0.25 mM), 10 μM primer 0.5 to 2.5 μl each (final concentration is 0.1 to 0.5 μM), template cDNA 0.1 to 0.5 μg, Ex-Taq polymerase ( In a reaction solution having a composition containing 1.25 units (Takara Bio Inc.), a three-step amplification cycle is repeated under the following conditions. First, as a denaturing step, for example, the temperature is kept at about 90 ° C. to about 96 ° C., preferably about 94 ° C. to about 95 ° C. for 1 minute to 15 minutes, preferably 1 minute to 2 minutes. Next, as the primer annealing step, for example, the temperature is kept at about 30 ° C. to about 70 ° C., preferably about 40 ° C. to about 60 ° C., for about 3 seconds to about 3 minutes, preferably about 5 seconds to about 2 minutes. . Further, as the extension step by DNA polymerase, for example, the temperature is kept at about 70 ° C. to about 75 ° C., preferably about 72 ° C. to about 73 ° C., for about 15 seconds to about 5 minutes, preferably about 30 seconds to about 4 minutes. Is called. This three-step amplification cycle is performed about 20 to about 50 times, preferably about 25 to about 40 times. The DNA fragment amplified by such PCR is purified by subjecting to low melting point agarose electrophoresis or the like, if necessary, and then recovered by ethanol precipitation, or, for example, QIAquick PCR Purification Kit (manufactured by Qiagen) It can be purified and recovered using a commercially available column for purifying DNA fragments. An appropriate solution such as water, physiological saline, 1 to 100 mM Tris-HCl, TE buffer [10 mM Tris-HCl, 1 mM EDTA, pH 7.4 to 8.0] is used as a solvent for dissolving the DNA fragments during recovery. Can be used.

  Examples of the probe used in the hybridization method include a polynucleotide comprising the base sequence represented by SEQ ID NO: 1, or a polynucleotide having a partial base sequence of the polynucleotide. As hybridization conditions, for example, 6 × SSC (0.9 M sodium chloride, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1 (w / v) Ficoll 400, 0.1 (w / v) polyvinylpyrrolidone, 0.1 ( w / v) BSA), 0.5 (w / v) SDS and 100 μg / ml denatured salmon sperm DNA, incubated at 65 ° C., then 1 × SSC (0.15 M sodium chloride, 0.015 M sodium citrate) and 0.5 Incubate twice for 15 minutes at room temperature in the presence of (w / v) SDS, and then in the presence of 0.1 × SSC (0.015M sodium chloride, 0.0015M sodium citrate) and 0.5 (w / v) SDS, 68 Examples include a condition of keeping at 30 ° C. for 30 minutes. In addition, for example, incubate at 65 ° C. in the presence of 5 × SSC, 50 mM HEPES pH 7.0, 10 × Denhardt's solution and 20 μg / ml denatured salmon sperm DNA, then incubate in 2 × SSC at room temperature for 30 minutes, and further in 0.1 × SSC In addition, there may be mentioned a condition in which the temperature is kept twice at 65 ° C. for 40 minutes. In addition, the gene of the present invention or the polynucleotide of the present invention is based on, for example, the phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984) based on the nucleotide sequence represented by SEQ ID NO: 1. It can also be prepared by performing chemical synthesis of nucleic acids according to the usual methods such as.

The gene of the present invention or the polynucleotide of the present invention obtained in this way is, for example, a gene described in Sambrook, J. et al., Molecular Cloning: A laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001), etc. They can be cloned into vectors according to engineering methods.
Specifically, for example, cloning can be performed using a commercially available plasmid vector such as TA cloning kit (Invitrogen) or pBluescriptII (Stratagene). The base sequence of the obtained gene of the present invention or the polynucleotide of the present invention is described in the Maxam Gilbert method (for example, Maxam, AM & W. Gilbert, Proc. Natl. Acad. Sci., USA, 74, 560, 1977, etc. And the Sanger method (for example, Sanger, F. & AR Coulson, J. Mol. Biol., 94, 441, 1975, Sanger, F. & Nicklen & AR Coulson, Proc. Natl. Acad. Sci., USA, 74, 5463, 1977, etc.). When an automatic DNA sequencer such as Model 377 manufactured by ABI is used, a corresponding DNA sequence kit such as Big Dye Terminator Cycle Sequencing Ready Reaction Kit manufactured by ABI can be used.

Next, the polynucleotide of the present invention used for detection of DNA or RNA will be described (hereinafter sometimes referred to as the present polynucleotide for detection of DNA etc.).
The polynucleotide of the present invention for detecting DNA or the like is a polynucleotide comprising any one of the following base sequences.
(1) Base sequence represented by base numbers 1274 to 1873 of the base sequence represented by SEQ ID NO: 1 (2) Specified by the base sequence represented by base numbers 1274 to 1873 of the base sequence represented by SEQ ID NO: 1 Arbitrary arbitrary base sequences selected from the region and having a base length of 20 to 60 (3) A base sequence complementary to any one of the base sequences described in (1) to (6) above, wherein As item number (2), for example,
(2a) a base sequence having an arbitrary continuous length of 60 bases selected from the region specified by the base sequence represented by base numbers 1274 to 1873 of the base sequence represented by SEQ ID NO: 1, (2b) sequence A base sequence having an arbitrary continuous 50 base length selected from the region specified by the base sequence represented by base numbers 1274 to 1873 of the base sequence represented by number 1, (2c) represented by SEQ ID NO: 1 A base sequence having an arbitrary continuous 35 base length selected from the region specified by the base sequence represented by base numbers 1274 to 1873 of the base sequence, (2d) a base of the base sequence represented by SEQ ID NO: 1 A base sequence having an arbitrary continuous 30 base length selected from the region specified by the base sequence represented by the numbers 1274 to 1873; (2e) represented by SEQ ID NO: 1 Selected from the region from nucleotide number 1274 of the base sequence specified by the nucleotide sequence represented by 1873, it can be mentioned nucleotide sequence or the like having an arbitrary 20 nucleotide long contiguous.

The polynucleotide of the present invention for detecting DNA or the like having the characteristics of any one of the above item numbers (1) to (3) is one to a plurality of polynucleotides used for detecting a desired gene to be analyzed. It can be selected by using the similarity between the polynucleotide and the property as an index. For example, in the case of using a DNA array that detects DNA or RNA using a long-chain cDNA as described later, from the base number 1274 of the base sequence represented by SEQ ID NO: 1 represented by item number (1) It is preferable to use a polynucleotide having a base sequence of 1873. In the case of detecting DNA or RNA by a DNA array using a short-chain polynucleotide or a method such as RT-PCR, any one of the polynucleotides shown in item number (2) or (3) Is preferably used. Specifically, as the item number (2), for example, starting from any one of the base numbers 1461 to 1478, 1595 to 1690, and 1768 to 1809 of the base sequence represented by SEQ ID NO: 1, Examples include base sequences having a length of 60 bases that end in base numbers 1520 to 1537, 1654 to 1749, and 1827 to 1868 of the base sequence shown. Further, for example, it starts from any one of base numbers 1274 to 1275, 1461 to 1487, 1595 to 1695, and 1773 to 1814 of the base sequence represented by SEQ ID NO: 1, and each base number 1323 of the base sequence represented by SEQ ID NO: 1 To 1524, 1510 to 1536, 1644 to 1744, and 1822 to 1863. Further, for example, it starts from any one of base numbers 1274 to 1282, 1461 to 1490, 1572 to 1702, 1780 to 1821 of the base sequence represented by SEQ ID NO: 1, and each base number 1308 of the base sequence represented by SEQ ID NO: 1 To 1316, 1495 to 1524, 1606 to 1736, 1814 to 1855, and a base sequence having a continuous length of 35 bases. Also, for example, starting from any one of base numbers 1274 to 1285, 1461 to 1493, 1576 to 1580, 1595 to 1705, 1783 to 1799, and 1817 to 1824 of the base sequence represented by SEQ ID NO: 1, Examples of the base sequence shown include base numbers 1303 to 1314, 1490 to 1522, 1605 to 1609, 1624 to 1734, 1812 to 1828, and 1846 to 1853. Also, for example, starting from any one of the base numbers 1332, 1335, 1336, 1348, 1349, 1353, 1382, 1448, 1449, 1533, 1621, 1724, 1726 of the base sequence represented by SEQ ID NO: 1, respectively, The base sequence having a length of 20 bases that ends with base numbers 1351, 1354, 1355, 1367, 1368, 1372, 1401, 1467, 1468, 1552, 1640, 1743, 1745 of the base sequence indicated by 1 is given. Can do. More specifically, properties such as sequence length and melting point are equivalent to one to a plurality of polynucleotides used for detecting a desired gene from the base sequences specified above. It is preferred to select a polynucleotide.
In addition, as indicated by item number (3), when DNA or RNA is detected using a hybridization method such as a DNA array or Northern method, either of the above item numbers (1) or (2) is used. It is preferable to use a polynucleotide having a base sequence complementary to the base sequence shown.
The polynucleotide characterized by having the base sequence shown in any of the above item numbers (1) to (3) can be used not only independently but also in a plurality of combinations. it can. In use as a primer or probe in the RT-PCR method described later, a polynucleotide having a base sequence represented by item numbers (2) to (3) (base length 20 to 60, preferably base length 20 to 50) Among these, it is preferable to select a suitable polynucleotide for carrying out the PCR reaction. Polynucleotides can be selected from the above-mentioned polynucleotides using commercially available gene analysis software such as DNASIS (manufactured by Hitachi Software). In order to increase the reliability at the time of detection, a polynucleotide that detects the base sequence region of a plurality of different genes is also used. In this case, the item number (2) or (3 It is desirable to use a plurality of polynucleotides each having a base sequence represented by () and having base sequences that do not overlap each other.

The present polynucleotide for detection of DNA or the like may be obtained by any method as long as it can be obtained.
For example, as specifically shown in the section of the obtained gene of the present invention or the polynucleotide of the present invention, it is obtained from a marmoset tissue such as a common marmoset or an animal-derived cultured cell using a hybridization method, a PCR method, or the like. In addition, based on the information of the base sequence, chemical synthesis with a commercially available DNA synthesizer can be mentioned as a preferred method.

Next, the composition of the present invention will be described.
The composition of the present invention is a composition for detecting DNA or RNA and is characterized by containing the polynucleotide of the present invention. The composition may be in a form in which the polynucleotide of the present invention is immobilized on a carrier. Among these forms, a representative one in which the polynucleotide of the present invention is immobilized on the surface of a solid substrate is a DNA array (hereinafter sometimes referred to as the DNA array of the present invention).
Gene expression analysis techniques are actively used in various aspects such as disease diagnosis, classification or occurrence prediction, or analysis of drug efficacy or toxicity / side effect mechanisms. As a gene expression analysis method, the Northern blotting method has been conventionally used. However, a quantitative reverse transcriptase-polymerase chain reaction method (hereinafter sometimes referred to as a quantitative RT-PCR method) or a DNA array. A method using s (hereinafter also referred to as a DNA array method) has been developed, and these methods are also currently used.
In the former gene expression analysis method (ie, quantitative RT-PCR method), polynucleotides such as mRNA or total RNA, which are transcription products of a desired gene, are usually extracted and purified independently from a plurality of test samples. After synthesizing the cDNA corresponding to the extracted and purified polynucleotide using reverse transcriptase, the amount of the synthesized cDNA is quantified using the PCR method, and the quantified amount of cDNA is measured for a plurality of test samples. The difference in the expression level of the desired gene is measured by relative comparison. The composition of the present invention is useful when performing a gene expression analysis technique involving the relative comparison.
In the latter gene expression analysis method (ie, DNA array method), polynucleotides such as mRNA or total RNA, which are transcription products of a desired gene, are usually extracted and purified independently from a plurality of test samples. -A labeled product in which cDNA corresponding to the purified polynucleotide or cRNA obtained by in vitro transcription reaction thereof was labeled with a fluorescent dye, radioisotope, etc. was hybridized to a DNA array. Thereafter, the amount of hybridized label is quantified based on fluorescence intensity, radiation intensity, etc., and the amount of cDNA or cRNA quantified is relatively compared among a plurality of test samples to determine the expression level of the desired gene. Measure the difference. The composition of the present invention is useful when performing a gene expression analysis technique involving the relative comparison.

The DNA array of the present invention includes a DNA array in which several to several hundred DNA probes are arranged depending on the number of DNA probes arranged on the surface of a solid substrate, and a DNA array in which a larger number of DNA probes are arranged. These are broadly divided into two types, but here both are included.
The DNA array of the present invention contains the polynucleotide of the present invention as a probe, and the number of other probes and the base sequence thereof are not particularly limited. For example, a DNA array containing the polynucleotide of the present invention as a probe and a probe corresponding to several to tens of thousands of genes other than the gene of the present invention can be mentioned. Preferably, a DNA array containing the polynucleotide of the present invention as a probe and a probe corresponding to a gene derived from several to tens of thousands of common marmoset other than the gene of the present invention can be mentioned.

The DNA array of the present invention comprises, for example, a DNA fragment consisting of all or part of the base sequence represented by SEQ ID NO: 1 in accordance with the method described in the Genome Function Research Protocol Experimental Medicine Supplement (Yodosha) and the like. It can be manufactured by both the steps of preparing a DNA probe to be included and immobilizing the prepared DNA probe on the surface of a solid substrate. In addition, based on the information on the base sequence of the DNA probe, as described in Fodor, SPA et al., Science, 251, 767 (1991), bases were sequentially added onto a solid substrate for chemical synthesis. It is also possible to directly manufacture the DNA array by moving. Hereinafter, the former production method will be described in detail, but the production method according to the DNA array of the present invention is not limited thereto.
First, a DNA probe is prepared. As a method for preparing a DNA probe, for example, it can be prepared by a PCR method or the like using a cDNA prepared from a common marmoset tissue as a template. Using the cDNA as a template, DNA is amplified by PCR using a DNA polymerase such as Ex Taq (Takara Bio). The DNA amplified in this manner may be inserted into a vector such as pUC118 and cloned. The base sequence of the DNA can be confirmed using a method according to the above Maxam Gilbert method, Sanger method or the like.

The amplified DNA after the PCR reaction can be used as a probe.
The amplified DNA after the PCR reaction is recovered by ethanol precipitation from a solution containing the DNA, or purified using a commercially available DNA fragment purification column such as QIAquick PCR Purification Kit (Qiagen). can do. As a solvent for dissolving the DNA in the purification, a suitable buffer such as dimethyl sulfoxide, water, physiological saline, 1 to 100 mM Tris-HCl, or a solution such as 3 × SSC can be used. Preferably, 3XSSC may be used. The concentration of DNA during lysis is adjusted to about 1 to about 100 μM. When DNA cloned into a vector is used, the target DNA fragment is excised with an appropriate restriction enzyme and purified on an agarose gel as necessary. The obtained DNA fragments can be separated into gels using a commercially available DNA fragment purification column such as QIAquick Gel Extraction Kit (Qiagen). The DNA fragment separated from the gel is dissolved in the above solvent. Alternatively, DNA for a DNA probe may be obtained by performing PCR again using a base sequence such as T7, T3, SP6 or the like that is usually set at both ends of the cloning region of the cloning vector as a primer.
When the length of the DNA fragment arranged on the DNA array is about 20 bases to about 100 bases, chemical synthesis using a commercially available DNA synthesizer based on the information of the base sequence can be mentioned as a preferred method. The chemically synthesized DNA fragment is purified by the above-described DNA fragment purification column, high performance liquid chromatography, or the like, and dissolved in the above solvent.

  Next, the DNA obtained above is immobilized on a carrier such as a solid substrate. Examples of the carrier include a solid substrate such as a glass plate, a quartz plate, a silicon wafer, and a membrane filter. The size of the carrier may be appropriately set according to the number of DNA probes arranged on the carrier, the size of the DNA probe spot, and the like. As a method for immobilizing DNA, an appropriate method may be selected according to the type of DNA, the type of carrier, and the like. For example, when DNA to be immobilized is cDNA, PCR amplification product, etc., it is electrostatically bound to a solid support surface-treated with polycation such as polylysine, polyethyleneimine, polyalkylamine, etc. using the charge of DNA. A method of covalently bonding DNA introduced with a functional group such as amino group, aldehyde group, SH group, biotin, etc. to a solid layer surface introduced with a functional group such as amino group, aldehyde group or epoxy group. Can be mentioned. In order to place the prepared DNA on a carrier, using a micropipette, pipetman, etc., a method in which the solution in which the DNA is dissolved is manually dropped, at a position indicated in advance in a size of several tens to several hundreds of μm. You may use the method of arrange | positioning by machine operation. As a DNA arrangement method, there are a pin method by mechanical contact of a carrier at the tip of a pin, an ink jet method applying the principle of an ink jet printer, a capillary method by a capillary tube, etc. Any method may be used. Examples of the pin method include Affymetrix 427 Arrayer (Affymetrix), and examples of the capillary method include Lucidea Array Spotter (Amersham Bioscience), but are not limited thereto. The amount of DNA solution to be arranged is preferably 0.01 to 100 nl in the case of mechanical arrangement, and 0.1 to 100 μl in the case of manual arrangement.

Next, the use of the present invention and the measurement method of the present invention will be described.
The present invention uses the test for correcting the transcript amount when measuring the difference in the expression level of a desired gene in two or more test samples as the difference in the transcript amount of the gene. It is the use of the polynucleotide of the present invention as an internal standard serving as a reference for the expression level of a gene in a sample. In the use of the present invention, the test sample may be a sample derived from a common marmoset.
The measurement method of the present invention is a method for measuring a difference in the expression level of a desired gene in two or more kinds of test samples as a difference in the amount of a transcription product of the gene, (1) A first step of measuring the amount of transcript of the 18S ribosomal RNA gene derived from common marmoset, (2) a second step of measuring the amount of transcript of the gene using a desired gene in the test sample, (3) In each test sample in two or more types of test samples, the amount of transcript of 18S ribosomal RNA gene measured in the first step in any test sample is compared with the first step in other test samples. A third step of calculating the relative ratio of the measured amount of 18S ribosomal RNA gene transcript, (4) the amount of the desired gene transcript measured in the second step A fourth step of correcting the transcript amount of the desired gene measured in the second step in each test sample in two or more test samples by multiplying by the relative ratio calculated in the third step; It is characterized by having. In the measurement method of the present invention, the test sample may be a sample derived from a common marmoset.

  As a method of measuring the amount of gene transcript in the first step and the second step in the measurement method of the present invention, any method can be used as long as it can quantitatively measure the amount of transcript. Northern hybridization method [Sambrook, J. et al., Molecular Cloning: A laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001)], quantitative RT-PCR method, DNA array method, in situ hybridization method, etc. Can be mentioned. Preferably, for example, a DNA array method or a quantitative reverse transcriptase-polymerase chain reaction method is used.

(1) Northern hybridization method First, DNA corresponding to the base sequence of a desired gene to be analyzed is prepared, and then a DNA consisting of all or part thereof is labeled to prepare a labeled probe. Here, the “labeled DNA probe” refers to a fluorescent dye, a radioisotope, etc. that are specifically hybridized to a gene having a specific base sequence and are used for the purpose of measuring the presence or absence and the expression level of the gene. It is a DNA fragment that is a label that has been labeled. Similarly, a labeled probe is prepared by labeling the DNA of the gene of the present invention or the polynucleotide of the present invention. The desired gene can be prepared by a PCR method or the like using a cDNA prepared from a common marmoset tissue by the above method as a template. Using the cDNA as a template, DNA is amplified by PCR using a DNA polymerase such as Ex Taq (Takara Bio). PCR conditions vary depending on the type of desired gene, the base sequence of the primer used, and the like, and examples include the reaction conditions described above. The amplified DNA may be cloned by inserting pUC118 or the like into a vector. The base sequence of the DNA can be confirmed using a method according to the above Maxam Gilbert method, Sanger method or the like.

A probe can be prepared by labeling the DNA of the desired gene thus prepared and the gene of the present invention or the polynucleotide of the present invention with a fluorescent dye, a radioisotope or the like as follows. For example, [α- ³² P] dCTP or [α- ³² P] dATP is contained using the DNA prepared as described above as a template and using an oligonucleotide having a partial sequence of the base sequence of the DNA as a primer. A probe labeled with ³² P can be obtained by performing PCR by adding dNTP to the reaction solution. Further, the DNA prepared as described above may be labeled using a commercially available labeling kit such as Random prime labeling Kit (Roche Diagnostics) or MEGALABEL (Takara Bio).

Next, Northern hybridization analysis is performed using the probe thus prepared. Specifically, in the second step of the measurement method of the present invention, total RNA or mRNA is prepared from two or more test samples derived from a common marmoset for which the amount of a desired gene transcript is to be measured. 20 μg of the prepared total RNA or 2 μg of mRNA was separated on an agarose gel, washed with 10 × SSC (1.5 M NaCl, 0.35 M sodium citrate), and then on a nylon membrane [for example, Hybond-N (Amersham Biosciences, etc.)] Transfer. Put the membrane in a polyethylene bag, hybridization buffer [6 x SSC (0.9 M NaCl, 0.21 M sodium citrate), 5 x Denhardt's solution [0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone , 0.1% BSA], 0.1% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, 50% formamide] and incubate at 50 ° C. for 2 hours, discard the hybridization buffer, Add 2 ml to 6 ml of hybridization buffer. Further, the labeled probe obtained by the above method is added and incubated at 50 ° C. overnight. In addition to the above, commercially available DIG EASY Hyb (Roche Diagnostics) can be used as the hybridization buffer. Remove the membrane and incubate in 50 ml to 100 ml of 2xSSC, 0.1% SDS at room temperature for 15 minutes, repeat the same procedure once, and finally in 50 ml to 100 ml of 0.1xSSC, 0.1% SDS. Incubate at 68 ° C for 30 minutes. By measuring the amount of label on the membrane, the amount of transcript of the gene can be measured.
Separately, total RNA or mRNA prepared from the same test sample is separated using an agarose gel by the above method and transferred to a nylon membrane. A transcription product of the gene of the present invention is obtained by adding a labeled probe corresponding to the gene of the present invention or the polynucleotide of the present invention obtained by the above method, performing hybridization according to the above conditions, and measuring the amount of label on the membrane. The amount can be measured.
In addition to separately preparing a nylon membrane to which total RNA or mRNA has been transferred, the labeled probe is removed from the nylon membrane that has measured the amount of transcript of the desired gene, and the amount of transcript of the gene of the present invention is measured. You can also In this case, for example, the hybridized labeled probe is removed by heating or boiling the membrane for 5 to 30 minutes in a solution containing 0.1 to 1% SDS. After returning the nylon membrane to room temperature, the amount of the transcription product of the gene of the present invention can be measured by performing hybridization with a labeled probe corresponding to the gene of the present invention or the polynucleotide of the present invention under the above-mentioned conditions.

(2) Quantitative RT-PCR
MRNA is prepared by the same method as described in (1) Northern hybridization method from two or more kinds of test samples derived from common marmoset for which the amount of transcript of the desired gene to be analyzed is to be measured. . For example, reverse transcriptase such as MMLV (Toyobo) is added to the prepared mRNA, and a reaction buffer [50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl ₂ , 75 mM KCl, 10 mM DTT] In the presence of 0.5 mM dNTP and 25 μg / ml oligo dT, the reaction is carried out at 42 ° C. for 15 minutes to 1 hour to prepare cDNA corresponding to the mRNA. In addition, you may prepare corresponding cDNA using a cDNA synthesis kit (Takara Bio).
PCR is carried out using the prepared cDNA as a template and DNA consisting of a part of the base sequence of the desired gene as a primer. Primers used for PCR may be designed using commercially available gene analysis software such as DNASIS (manufactured by Hitachi Software). For example, an oligonucleotide having a length of about 15 bases to about 50 bases, which is difficult to form an unnecessary secondary structure and has a highly specific base sequence for the gene can be used. . As PCR conditions, for example, Ex taq (Takara Bio) is used, and 2.5 mM dNTP in the reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ] and [ In the presence of α ³² P] -dCTP, for example, 94 ° C., 30 seconds, then 40 ° C. to 60 ° C., 2 minutes, 72 ° C., and 2 minutes of heat insulation can be mentioned as conditions for 25 to 55 cycles. it can. By subjecting the amplified DNA to polyacrylamide gel electrophoresis and measuring the radiation intensity of the separated DNA, the amount of the transcription product of the gene can be measured.
Also, for example, using Ex taq (Takara Bio), SYBR Green PCR Reagents (Applied Biosystems) in reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ] ) Prepare a 50 μl reaction solution containing 25 μl, and incubate at 50 ° C. for 2 minutes then 95 ° C. for 10 minutes using ABI7700 (Applied Biosystems), then 95 ° C. for 15 seconds then 60 ° C. for 1 minute PCR is carried out under the condition that the incubation is performed for 40 cycles. By measuring the fluorescence intensity of the amplified DNA, the amount of the transcription product of the gene can be measured.
PCR is performed using the cDNA as a template and the polynucleotide of the present invention as a primer. The PCR may be performed at the same time as PCR for the purpose of measuring the amount of transcript of a desired gene or at another time, but the same time is desirable.
Primers used for PCR may be designed using commercially available gene analysis software such as DNASIS (manufactured by Hitachi Software). For example, an oligonucleotide having a length of about 15 bases to about 50 bases, which is difficult to form an unnecessary secondary structure, a primer having a highly specific base sequence for the gene can be used. . The PCR conditions are preferably the same as those used for measuring the amount of transcript of the desired gene. For example, using Ex taq (Takara Bio), 2.5 mM dNTP and [α ³² P] -dCTP in reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ] In the presence, for example, 94 ° C., 30 seconds, then 40 ° C. to 60 ° C., 2 minutes, further 72 ° C., and 2 minutes of incubation can be mentioned as one cycle. By subjecting the amplified DNA to polyacrylamide gel electrophoresis and measuring the radiation intensity of the separated DNA, the amount of the transcription product of the gene of the present invention can be measured. Also, for example, using Ex taq (Takara Bio), SYBR Green PCR Reagents (Applied Biosystems) in reaction buffer [10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ] ) Prepare a 50 μl reaction solution containing 25 μl, and incubate at 50 ° C. for 2 minutes then 95 ° C. for 10 minutes using ABI7700 (Applied Biosystems), then 95 ° C. for 15 seconds then 60 ° C. for 1 minute PCR is carried out under the condition that the incubation is performed for 40 cycles. By measuring the fluorescence intensity of the amplified DNA, the amount of the transcription product of the gene of the present invention can be measured.

(3) DNA array analysis In order to measure the amount of the transcript of the desired gene to be analyzed and the amount of the transcript of the gene of the present invention in the test sample, a DNA array based on ordinary techniques may be used. . In such a DNA array, several to tens of thousands of DNA probes are arranged in an array on the surface of a solid substrate such as a membrane filter or a slide glass. By using this, the presence / absence and expression level of a desired gene in a test sample can be examined at a time. The DNA probe used here, like that used in the Northern hybridization method of (1) above, specifically hybridizes to a gene having a specific base sequence, and whether or not the gene is expressed and its expression. It is a DNA fragment used for the purpose of measuring the amount. Depending on the number of DNA probes arranged on the surface of the solid substrate, the DNA array includes a DNA array in which several to several hundred DNA probes are arranged and a DNA array in which a larger number of DNA probes are arranged. Although broadly classified into two types, both are included here.
The DNA array used in the present invention can be produced, for example, according to the method described in the Genome Function Research Protocol Experimental Medicine Supplement (published by Yodosha). Also, based on the information on the base sequence of the DNA probe, as described in Fodor, SPA et al., Science, 251, 767 (1991), bases were sequentially added onto a solid substrate for chemical synthesis. It is also possible to directly manufacture the DNA array by moving. Details of the DNA array production method are as described above, for example.

Hereinafter, an example of a method for measuring the amount of a transcript of the gene of the present invention using a DNA array will be described.
(3-1) Quantification by DNA macroarray (part 1)
MRNA is prepared from a test sample to be measured for the amount of transcript of the desired gene to be analyzed by the same method as described in the Northern hybridization method (1) above. For example, reverse transcriptase such as MMTV (Toyobo) is added to the prepared mRNA, and a reaction buffer [for example, 50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl ₂ , 75 mM KCl and 10 mM] In a solution containing DTT], labeled cDNA is prepared by reacting at 42 ° C. for 15 minutes to 1 hour in the presence of 0.5 mM dNT [α ³² P] -dCTP, P and 25 μg / ml oligo dT. At this time, a cDNA synthesis kit (Takara Bio) or the like may be used. A DNA array prepared by spotting a DNA probe having a base sequence that matches all or part of the base sequence of the desired gene to be analyzed, the gene of the present invention or the full-length cDNA of the present invention on a membrane filter is a polyethylene bag. In the hybridization buffer [6 × SSC (0.9 M NaCl, 0.21 M sodium citrate), 5 × Denhardt's solution [0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1 % BSA], 0.1% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, 50% formamide], and incubate at 50 ° C. for 2 hours. Add 2 ml to 6 ml of hybridization buffer. Further, the above DNA probe is added to this and incubated at 50 ° C. overnight. In addition to the above, a commercially available DIG EASY Hyb (Roche Diagnostics) can also be used as the hybridization buffer. After hybridization, the DNA array is taken out, immersed in 50 ml to 100 ml of 2 × SSC, 0.1% SDS, and incubated at room temperature for about 15 minutes. Furthermore, the same operation is repeated once, and finally, it is incubated in 50 ml to 100 ml of 0.1 × SSC, 0.1% SDS at 68 ° C. for 30 minutes. Next, by measuring the amount of label on the DNA array, the amount of the desired gene to be analyzed and the transcript of the gene of the present invention can be measured.

(3-2) Quantification by DNA microarray (part 2)
MRNA is prepared from a test sample to be measured for the amount of transcript of the desired gene to be analyzed by the same method as described in the Northern hybridization method (1) above. For example, reverse transcriptase such as MMTV (Toyobo) is added to the prepared mRNA, and a reaction buffer [for example, a solution containing 50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl ₂ , 75 mM KCl, and 10 mM DTT] In the presence of 0.5 mM dNTP, Cy3-dUTP (or Cy5-dUTP) and 25 μg / ml oligo dT, the reaction is performed at 42 ° C. for 15 minutes to 1 hour. Add an alkaline buffer (for example, a solution containing 1N NaOH and 20 mM EDTA), incubate at 65 ° C for 10 minutes, and remove the free Cy3 or Cy5 using Qiaquick PCR Purification kit (Qiagen). Prepare labeled DNA. The prepared fluorescently labeled DNA is subjected to hybridization and subsequent treatment to the DNA array by the same method as that described in (3-1) Quantification with DNA Array (Part 1). By measuring the amount of label on the DNA array with a scanner or the like, the amount of the desired gene to be analyzed and the transcript of the gene of the present invention can be measured.

(4) In-situ hybridization method Basically, it consists of (a) tissue fixation, embedding and section preparation, (b) probe preparation, (c) detection by hybridization. Other than using labeled RNA or DNA as a probe, for example, Heiles, H. et al., Biotechniques, 6, 978, 1988, Genetic Engineering Handbook Yodosha 278 1991, Cell Engineering Handbook, Yodosha, 214 1992, Cell Engineering Handbook, Yodosha, 222, 1992, and the like.
When preparing an RNA probe, first, for example, the desired gene to be analyzed and the gene of the present invention or the full-length cDNA are obtained by the same method as described in the Northern hybridization method of (1) above. Then, the DNA is incorporated into a vector having SP6, T7 or T3 RNA polymerase promoter (for example, Bluescript from Stratagene, pGEM from Promega, etc.) and introduced into E. coli to prepare plasmid DNA. Next, the plasmid DNA is cleaved with a restriction enzyme so that sense (for negative control) and antisense (for hybridization) RNA can be produced. Using both plasmid DNAs as templates, α- ³⁵ S-UTP etc. for radioactive labeling, digoxigenin UTP or fluorescein modified UTP as a substrate for non-radioactive labeling, and RNA using SP6, T7, T3 RNA polymerase After labeling during synthesis, RNA labeled in advance with a radioactive or non-radioactive substance is prepared by cleaving to a size suitable for hybridization by alkaline hydrolysis. As a kit based on these methods, for example, an RNA labeling kit (Amersham Bioscience) is commercially available for radiolabeling, and a DIG RNA labeling kit (Roche Diagnostics) is used for non-radioactive labeling. RNA color kits (Amersham Biosciences) are commercially available. When preparing a DNA probe, for example, a radioactive nucleotide labeled with ³² P or the like, or a nucleotide labeled with biotin, digoxigenin or fluorescein is converted into a nick translation method [Sambrook, J. et al., Molecular Cloning. : A laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001)] or random prime method [Feinberg, AP, B. Vogelstein, Anal. Biochem., 132, 6 (1083), Feinberg, AP, B. Vogelstein, Anal Biochem., 137, 266 (1984)] to prepare DNA previously labeled with a radioactive or non-radioactive substance. As kits based on these methods, for example, nick translation kits (Amersham Biosciences) and Random Prime Labeling Kit (Roche Diagnostics) are commercially available for radioactive labeling, and for non-radioactive labeling. DIG DNA labeling kits (Roche Diagnostics), DNA color kits (Amersham Biosciences), etc. are commercially available.
Specifically, the target gene to be analyzed and the test sample to be measured for the amount of the transcription product of the gene of the present invention are fixed with paraformaldehyde and embedded in paraffin, and then sliced sections are prepared. Paste this on the slide glass. In addition, the test sample is frozen in liquid nitrogen to prepare a microthin layer section, which is attached to a slide glass. In this way, a slide glass section is obtained.
Next, in order to remove substances that are present in the test sample and react nonspecifically with the probe to be used, the slide glass section prepared as described above is treated with proteinase K, and acetylated. Turn into. Next, hybridization between the slide glass section and the probe prepared as described above is performed. For example, after heating the probe at 90 ° C. for 3 minutes, a solution obtained by diluting the probe with a hybridization solution is dropped onto the slide glass section. After dropping, the slide glass slice is covered with a film, and kept in a moisture chamber at 45 ° C. for 16 hours to form a hybrid. After hybridization, nonspecific adsorption or unreacted probe is washed away (when RNA probe is used, RNase treatment is also added). By measuring the amount of label on the slide glass section, the amount of the desired gene to be analyzed and the transcript of the gene of the present invention can be measured.

Next, in the third step of the measurement method of the present invention, in each test sample in two or more types of test samples, the amount of transcript of 18S ribosomal RNA gene measured in the first step in any test sample, The relative ratio of the transcript amount of the 18S ribosomal RNA gene measured in the first step in other test samples is calculated.
Specifically, for example, n test samples were measured, and the amount of the transcript of the 18S ribosomal RNA gene derived from the common marmoset in the xth (x ≦ n) test sample was defined as Sx, and the i th ( When the amount of transcript of the 18S ribosomal RNA gene derived from the common marmoset in the test sample of i ≦ n) is Si and the calculation is performed based on the Si, the relative ratio Cx is expressed by the following equation.
Cx = Si / Sx
Here, the “arbitrary test sample” may be any test sample of the measured test samples, but if a test sample that serves as a control is used for the measurement, Is preferably used.

In the fourth step of the measurement method of the present invention, each test in two or more types of test samples is performed by multiplying the amount of transcript of the desired gene measured in the second step by the relative ratio calculated in the third step. The amount of transcript of the desired gene measured in the second step on the sample is corrected.
Specifically, for example, n test samples were measured, and the amount of transcript of a desired gene to be analyzed in the x th (x ≦ n) test sample is Tx, The relative ratio of the transcript amount of the 18S ribosomal RNA gene derived from the common marmoset in the x-th test sample to the i-th (i ≦ n) test sample calculated in the process is Cx, and the i-th test sample 18S. When calculating based on the transcription product amount of the ribosomal RNA gene, the transcription product amount ATx of the desired gene to be analyzed corrected by this step is expressed by the following equation.
ATx = Tx ・ Cx

  The following examples illustrate the invention in more detail, but the invention is not limited to these specific examples.

Example 1 (Cloning of 18S ribosomal RNA gene derived from common marmoset)
(1) Preparation of total RNA Total RNA was extracted from the liver of a common marmoset (available from CLEA Japan).
A 47-month-old male common marmoset was dissected and the liver was excised, and then the excised liver was subdivided with tweezers and scissors, and immediately immersed in RNAlater (Ambion) at room temperature overnight. After standing, it was stored at −20 ° C. until just before use. 10 ml of TRIZOL reagent (manufactured by Invitrogen) was added to 1 g of wet weight of the preserved liver tissue, homogenized with a Polytron homogenizer while cooling with ice, and left at room temperature for 5 minutes. Next, this was centrifuged (4 ° C., 9,000 rpm, 10 minutes), and then the aqueous layer was recovered in a 50 ml centrifuge tube (Asahi Techno Glass). To the recovered aqueous layer, 1/5 volume of chloroform (manufactured by Kanto Chemical Co., Inc.) of TRIZOL reagent was added. The resulting mixture was vigorously stirred up and down for 15 seconds and then allowed to stand at room temperature for 5 minutes. This was then centrifuged (4 ° C., 9,000 rpm, 10 minutes), and the aqueous layer was collected in a new 50 ml centrifuge tube. To the collected aqueous layer, 1/2 volume of 2-propanol (manufactured by Kanto Chemical) of TRIZOL reagent was added. The resulting mixture was mixed by inversion and then allowed to stand at room temperature for 10 minutes. After standing, this was centrifuged (4 ° C., 9,000 rpm, 10 minutes), and then the supernatant was removed to obtain an RNA pellet. The obtained pellet was dissolved in 1 ml of DEPC-treated sterilized distilled water (manufactured by Nacalai Tesque). To 1 ml of the RNA solution thus obtained, 3.8 ml of RLT buffer (10 μl β-mercaptoethanol / mL RLT buffer) attached to RNeasy Midi Kit (Qiagen) was added and mixed. Furthermore, 2.8 ml ethanol (manufactured by Kanto Chemical) was added to the mixture and mixed. The mixture was divided into equal parts, and applied to two RNeasy spin columns attached to the RNeasy Midi Kit, followed by centrifugation (room temperature, 10,000 rpm, 15 seconds). After centrifugation, the eluate was again applied to the same RNeasy spin column and centrifuged (room temperature, 10,000 rpm, 15 seconds). After centrifugation, the eluate was discarded, 2.5 ml each of RPE buffer attached to RNeasy Midi Kit diluted 4-fold with ethanol was applied, and centrifuged (room temperature, 10,000 rpm, 15 seconds). After centrifugation, the eluate was discarded again, and 2.5 ml each of RPE buffer diluted with ethanol was applied and centrifuged (room temperature, 14,000 rpm, 2 minutes). After centrifugation, the column was transferred to a new Eppendorf tube, 0.25 ml each of distilled water attached to the RNeasy Midi Kit was added, and the mixture was allowed to stand at room temperature for 1 minute. After standing, this was centrifuged (room temperature, 10,000 rpm, 1 minute) to obtain total RNA as its eluate.

(2) Preparation of cDNA 20 μl of a mixed solution containing 6 μg of the total RNA prepared in (1) and 1 μg of the primer having the base sequence shown in SEQ ID NO: 4 was heated at 70 ° C. for 10 minutes and then cooled on ice. did. After cooling, the mixture is 8 μl of 5 × First Strand cDNA Buffer contained in SuperScript II Choice System for cDNA Synthesis (Invitrogen), 4 μl of 0.1 M DTT contained in the kit, and 2 μl of 10 mM dNTPMix contained in the kit, DEPC treatment After adding 4 μl of water and 2 μl of Super ScriptII RT contained in the kit, the mixture was heated at 37 ° C. for 2 hours, further heated at 75 ° C. for 5 minutes, and then the mixture was cooled on ice to obtain cDNA. The cDNA thus obtained was stored at −20 ° C. until use after cooling.

(3) Amplification of the gene of the present invention by PCR method Using the cDNA obtained above as a template, a forward primer having the base sequence represented by SEQ ID NO: 2, a reverse primer having the base sequence represented by SEQ ID NO: 3, and Ex Amplified DNA was obtained by performing PCR using Taq polymerase (manufactured by Takara Bio). The PCR reaction solution composition was 1 μl of the above cDNA, 5 μl of 10 × PCR buffer attached to Ex taq polymerase (Takara Bio), 4 μl of 2.5 mM dNTP attached to Ex taq polymerase (Takara Bio), The above primers at a concentration of 10 μM were each 1 μl, Ex taq polymerase 0.5 μl, and sterilized purified water 37.5 μl. The PCR reaction is first performed at 94 ° C for 2 minutes, followed by reaction conditions of 30 cycles of 94 ° C for 30 seconds, then 60 ° C for 30 seconds, and then 72 ° C for 1 minute. It was. The reaction mixture was further warmed at 72 ° C. for 5 minutes and then stored at 4 ° C. 50 μl collected from the reaction mixture was subjected to agarose gel electrophoresis, and a band showing the mobility corresponding to the amplified DNA was excised and purified using a QIAquick Gel Extraction Kit (Qiagen). Recovered in sterile purified water.

(4) Preparation of gene clone of the present invention The amplified DNA obtained in (3) above was cloned using a TA vector.
5 μl of the solution containing the amplified DNA and 1 μl of 50 mg / ml pGEM-T vector (Promega) were mixed, and both were reacted using DNA Ligation Kit Ver.2 (Takara Bio). Escherichia coli DH5α competent cells were transformed with the obtained reaction solution. Colonies were collected using the absence of staining for betagalactosidase using ampicillin resistance, IPTG and X-gal as indicators, and then plasmid DNA was prepared from the collected colonies. The base sequence of the prepared plasmid DNA was determined by the dye terminator method using an ABI model 3700 auto sequencer (Applied Biosystems). As a sequencing primer, an oligonucleotide having a base sequence represented by SEQ ID NOs: 5 to 8 chemically synthesized by a DNA synthesizer was used. The obtained nucleotide sequence was edited and aligned using DNA sequence analysis software DNASIS (manufactured by Hitachi Software Co., Ltd.) to obtain the nucleotide sequence represented by SEQ ID NO: 1. When the homology search of the nucleotide sequence obtained in this way was performed with respect to the NCBI gene sequence which is a public database using the BLAST method, the nucleotide sequence of the human 18S ribosomal RNA having the registration number X03205 was found. It was found that the homology was high, and it was confirmed to be an 18S ribosomal RNA gene derived from a common marmoset.

(5) Confirmation of expression of the gene of the present invention using RT-PCR method In order to confirm the amount of transcripts in each tissue of the 18S ribosomal RNA gene derived from common marmoset cloned in (4) above, various differences were observed. RNA was extracted from individuals and different types of tissues, and the expression of mRNA was examined using RT-PCR.
Tissues were excised from 4 common marmoset (obtained from CLEA Japan, Inc.), 47 to 132 months old, and total RNA was extracted using the method described in (1) above. Extracted tissues are liver, lung, heart, skeletal muscle, brain and kidney (above, individual number 1), liver, heart and skeletal muscle (above, individual number 2), liver and lung (above, individual number 3), There are 12 types of brain (individual number 4). Next, 12 μl of a mixed solution containing 1 μg of each of the total RNA and 1 μg of d (T) _12-18 primer (manufactured by Amersham Biosciences) was heated at 70 ° C. for 10 minutes and then cooled on ice. After cooling, the mixture contains 4 μl of 5 × First Strand cDNA Buffer contained in SuperScript II Choice System for cDNA Synthesis (Invitrogen), 2 μl of 0.1M DTT contained in the kit, 1 μl of 10 mM dNTPMix contained in the kit, and the kit After adding 1 μl of Super ScriptII RT contained in the mixture, the mixture was heated at 42 ° C. for 60 minutes, further heated at 75 ° C. for 15 minutes, and then the mixture was cooled at 4 ° C. to obtain cDNA.
The cDNA thus obtained was diluted by adding 80 μl of purified water and stored at −20 ° C. until use. Using the cDNA thus obtained as a template, a forward primer having the base sequence represented by SEQ ID NO: 9, a reverse primer having the base sequence represented by SEQ ID NO: 10, and Ex Taq polymerase (manufactured by Takara Bio Inc.) was used. RT-PCR was performed. The RT-PCR reaction composition is 3 μl of the diluted cDNA solution, 3 μl of 10 × PCR buffer attached to Ex taq polymerase (Takara Bio), and Ex taq polymerase (Takara Bio). The above primers with a concentration of 2.5 mM dNTP 2.4 μl and 10 μM were 0.6 μl, Ex taq polymerase 0.5 μl, and sterilized purified water 22.6 μl, respectively. The PCR reaction is first performed at 94 ° C for 2 minutes, followed by reaction conditions of 30 cycles of 94 ° C for 30 seconds, then 60 ° C for 30 seconds, then 72 ° C for 30 seconds. It was. The reaction mixture was further warmed at 72 ° C. for 5 minutes and then stored at 4 ° C. 3 μl collected from the reaction mixture was subjected to agarose gel electrophoresis containing ethidium bromide.
As a result, as shown in FIG. 1, bands emitting even fluorescence were detected in 12 types of samples subjected to electrophoresis. From this result, it was confirmed that all 12 types of tissues subjected to electrophoresis contained almost the same amount of 18S ribosomal RNA gene mRNA regardless of the type of individual or tissue.

  According to the present invention, an 18S ribosomal RNA gene derived from a common marmoset, use thereof, and the like can be provided.

It is the result of RT-PCR which shows the expression distribution in each structure | tissue of the common marmoset of the 18S ribosomal RNA gene derived from a common marmoset. In the figure, “1”, “2”, “3”, and “4” indicate individual numbers from which each tissue was extracted (Example 5).

SEQ ID NO: 1
Common marmoset-derived 18S ribosomal RNA gene SEQ ID NO: 2
Oligonucleotide primer SEQ ID NO: 3 designed for PCR
Oligonucleotide primer designed for PCR SEQ ID NO: 4
Oligonucleotide primer designed for cDNA synthesis SEQ ID NO: 5
Oligonucleotide primer SEQ ID NO: 6 used for DNA sequencing
Oligonucleotide primer SEQ ID NO: 7 used for DNA sequencing
Oligonucleotide primer SEQ ID NO: 8 used for DNA sequencing
Oligonucleotide primer SEQ ID NO: 9 used for DNA sequencing
Oligonucleotide primer designed for RT-PCR SEQ ID NO: 10
Oligonucleotide primers designed for RT-PCR

Claims (10)

  18S ribosomal RNA gene derived from common marmoset. A polynucleotide comprising an 18S ribosomal RNA gene derived from a common marmoset or a partial fragment thereof, wherein the polynucleotide has any of the following base sequences:
(1) The base sequence represented by SEQ ID NO: 1 (2) The base sequence having 95% or more base identity with respect to the base sequence represented by SEQ ID NO: 1 (3) As described in (1) to (2) above A base sequence complementary to any base sequence (4) A partial base sequence in any one of the base sequences described in (1) to (3) above A polynucleotide comprising any one of the following base sequences:
(1) Base sequence represented by base numbers 1274 to 1873 of the base sequence represented by SEQ ID NO: 1 (2) Specified by the base sequence represented by base numbers 1274 to 1873 of the base sequence represented by SEQ ID NO: 1 Arbitrary arbitrary base sequence selected from the region and having a base length of 20 to 60 (3) A base sequence complementary to any one of the base sequences described in (1) to (6) above   A composition for detecting DNA or RNA, comprising the polynucleotide according to claim 2 or 3.   The composition according to claim 4, wherein the polynucleotide according to claim 2 or 3 is immobilized on a carrier.   Expression of a gene in the test sample for correcting the transcription product amount when measuring the difference in the expression level of a desired gene in two or more test samples as a difference in the transcription product amount of the gene Use of the polynucleotide according to claim 2 or 3 as an internal standard for determining the amount.   The use of the polynucleotide according to claim 6, wherein the test sample is a sample derived from a common marmoset. A method for measuring a difference in the expression level of a desired gene in two or more types of test samples as a difference in the transcription level of the gene,
(1) A first step of measuring the amount of transcript of 18S ribosomal RNA gene derived from common marmoset using the polynucleotide according to claim 2 or 3,
(2) a second step of measuring the amount of a transcription product of the gene using a desired gene in the test sample;
(3) In each test sample in two or more types of test samples, the amount of transcript of 18S ribosomal RNA gene measured in the first step in any test sample is compared with the first step in other test samples. A third step of calculating the relative ratio of the transcript amount of the measured 18S ribosomal RNA gene;
(4) By multiplying the amount of transcript of the desired gene measured in the second step by the relative ratio calculated in the third step, the second step in each test sample in two or more types of test samples A fourth step of correcting the measured transcript amount of the desired gene,
A measuring method characterized by comprising:   The measurement method according to claim 8, wherein the test sample is a sample derived from a common marmoset. The method for measuring the amount of a gene transcript in the first step and / or the second step is any one of a DNA array method and a quantitative reverse transcriptase-polymerase chain reaction method. The measuring method according to 8 or 9.

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