JPH07231797A - Method for determining mrna using rt-pcr method - Google Patents

Abstract

PURPOSE:To accurately determine an amount of mDNA by directly comparing and determining cDNA derived from mRNA of target gene with chromosome DNA. CONSTITUTION:A cDNA synthesized from mRNA to be detected derived from a part not containing intron of a gene is amplified together with a chromosome DNA of a part corresponding to the cDNA. Thereby, all of mRNA extracted from a sample can be used as a sample for PCR without requiring previously preparing mRNA in which point mutation is introduced or without requiring measuring the weight of the sample or an amount of total mRNA.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying mRNA in a tissue, and more particularly to a gene diagnostic method using a gene amplified by a gene (DNA) amplification method.

[0002]

2. Description of the Related Art Accurate measurement of the expression level of individual chromosomal genes in tissues or cells gives extremely important information for diagnosing pathological conditions and determining drug effects. Accurate quantification of mRNA in tissues has been difficult due to the low abundance and low quantification of the PCR method.

Conventionally, mRNA is measured by the gene amplification method (Polymerase Chain Reaction method, hereinafter abbreviated as PCR method) shown by Curry Banks Maris; JP-A-61-274697 and JP-A-62-281. The method was generally practiced. This is a gene that is constitutively expressed in a cell having a completely different base sequence from a specific gene in a tissue (for example, various hormone genes or various hormone receptor genes) and a specific gene of interest. (For example, β-actin gene)
A is simultaneously reverse-transcribed to produce each cDNA, which is amplified and the relative expression level in cells is comparatively quantified from the ratio of the two (RT-PCR method; Daniel A. Lavorie. Et al., Science 241, 70
8 to 712, 1988). In this method, since the base sequences of two mRNAs whose expression levels are compared are different from each other, when cDNA is synthesized by using a reverse transcriptase, the efficiency of producing cDNA is different, and further, when the prepared cDNA is amplified by PCR. However, there is a problem in that the amplification efficiency is different, and there is a problem in quantification. Therefore, this method could only confirm the presence or absence of expression or compare the relative expression levels.

The following has been implemented as an improved method of this method. That is, the mRNA of the target gene is separately prepared in advance. This mRNA is inserted (or removed) with a restriction enzyme site that does not exist (or exists) by point mutation so that it can be distinguished from the target mRNA. When this is added to a sample and simultaneously subjected to PCR, the gene sequences differ only in one place, and therefore the amplification efficiency may be considered to be the same. For this reason,
Quantitative performance is improved (Mikle Becker-Andre et al.
Nucleic Acid Research, Vol. 17, 94
37-9446, 1989). However, in the above-mentioned improved method, it is necessary to prepare in advance mRNA into which a point mutation corresponding to the target gene has been introduced. Further, it is necessary to measure the weight of the sample or the amount of extracted total mRNA separately, and there is a drawback that the target amount of mRNA in the tissue cannot be accurately quantified unless the sample is large.

[0005]

On the other hand, in the method of the present invention, point mutation-introduced mRNA is prepared in advance by directly comparing and quantifying the cDNA derived from the mRNA of the target gene with the chromosomal DNA. There is no need to measure the weight of the sample or the amount of extracted total mRNA separately, and the mRN extracted from the sample is unnecessary.
It is possible to use all A as PCR samples. That is, in the method of the present invention, a cDNA synthesized from an mRNA to be detected derived from a portion not containing an intron of a gene is a chromosomal DNA of a portion corresponding to the cDNA.
A method for quantifying mRNA by RT-PCR, which is characterized in that amplification is carried out together with, and more specifically, 1) synthesis of cDNA from mRNA to be detected derived from a portion not containing intron of gene is synthesized. 2) cDNA synthesized in step 1), which is carried out using an RT primer selected so as to introduce a new restriction enzyme site not included in the portion or to remove the restriction enzyme site included in the portion to be synthesized, PCR is carried out using a set of PCR primers having a nucleotide sequence capable of coamplifying the chromosomal DNA of the portion corresponding to the cDNA, and PCR containing the RT primer sequence portion of step 1).
A product is obtained, 3) the PCR product amplified in step 2) is treated with a restriction enzyme capable of cleaving the site introduced or removed in step 1), and 4) the PCR product treated in step 3) is electrophoresed. PCR product derived from cDNA and PC derived from chromosomal DNA
It is a method including each step of separating the R product and 5) quantifying each PCR product to quantify the mRNA to be detected.

The method of the present invention is described in detail below.
A feature of the invention is that the
It is to detect the expression level of mRNA specifically and quantitatively by simultaneously amplifying the cDNA derived from NA and the chromosomal DNA by the PCR method. That is, select a part that does not contain an intron of a specific gene,
CDNA is synthesized with complementary RT primers with base substitutions so as to create new restriction enzyme sites or to remove existing restriction enzyme sites. As a result, a cDNA in which a restriction enzyme site that originally did not exist in the chromosomal DNA that is simultaneously amplified by PCR described below is introduced or a restriction enzyme site that was present is removed is synthesized. In this way, the cDNA into which the new restriction enzyme site has been introduced or removed and the original chromosomal DNA are replaced with bases so that the new restriction enzyme site of the RT primer described above is generated or removed. 5 '
PCR product using two primers, a short primer on the side and a complementary primer on the 3'side of the cDNA, and a PCR product into which a new restriction enzyme site derived from cDNA and a restriction enzyme site derived from chromosomal DNA were introduced. Or a PCR product containing no restriction enzyme site derived from cDNA and a PCR product containing a restriction enzyme site derived from chromosomal DNA are synthesized. These two PCs
Since the R products are synthesized by the same set of PCR primers, the ratios of synthesis are considered to be exactly the same. Thereby, the difference in amplification efficiency at the PCR stage can be excluded. Then, the amplified PCR product is treated with the above-mentioned restriction enzyme to obtain a cDNA having a new restriction enzyme site introduced therein.
In the case of A, only the PCR product derived from mRNA, or the cDNA derived from the chromosome when the restriction enzyme site is removed
Only is cut into two short fragments. These PCR products can be separated into two, by performing electrophoresis, into a long PCR product derived from a chromosome and a short PCR product derived from mRNA, or a short PCR product derived from a chromosome and a long PCR product derived from mRNA. it can. Separated PCR products can be detected by ethidium bromide staining or by hybridizing with radiolabeled or fluorescently labeled probes.
The concentration of the detected band can be quantified visually or by a densitometer. The gene in the chromosome for the purpose of quantification by this method is always constant except when gene amplification such as oncogene occurs, and the degree of expression can be quantified by comparison with chromosomal DNA. . The outline of the method of the present invention is shown in FIG.

<Study on Quantitativeness> Male I of 5 to 7 weeks of age
In the DNA extracted from the kidney of the CR mouse, the RT primer shown in SEQ ID NO: 1 and the P shown in SEQ ID NOS: 2 and 3 were used.
Of the CR primers, the sense side primers (each 20
0 nM) and 1.25 U of Taq polymerase are added to bring the volume to 50 μl with 10 mM Tris buffer (pH 8.3) containing 50 mM NaCl, 4 mM MgCl ₂ , 1 mM DTT, 0.2 mM each of dNTP and 0.01% gelatin. . After heating at a denaturation temperature of 94 ° C for 40 seconds,
Forty cycles of PCR were performed with heating at an annealing temperature of 60 ° C for 30 seconds and then extension temperature of 72 ° C for 10 seconds. As a result, a 206 base pair mutant DNA in which the 248th guanine of the prostaglandin E2 receptor gene was replaced with adenine was obtained. This was purified by acrylamide gel electrophoresis. Similarly, using the PCR primer shown in SEQ ID NO: 2, a 206 base pair wild type DNA was obtained in the same manner as above. Wild type DNA
And the mutant DNA were mixed at a constant ratio and PCR was performed in the same manner as above. 10 μCi of ³⁵ S when performing PCR
Labeled dCTP was added. 150 mM KCl, 10 mM MgC
P of 10μl consisting of Tris buffer 12mM containing l ₂
Add 2 μl of MboI (1 u / μl) to the CR product,
Incubated for 2 hours at ° C. PC treated with MboI
R products are separated by acrylamide gel electrophoresis,
After drying the gel, it was autoradiographed and quantified with a densitometer. As shown in the figure, the ratio of wild-type DNA to mutant DNA was 1: 0.125 to 1: 3.
It showed extremely good quantification in the range (Figs. 2 and 3).

[0008]

EXAMPLES The method of the present invention will be described below with reference to examples. The base sequences of the primers used in the following examples are
It is as shown in the sequence listing. SEQ ID NO: 1 is the base sequence of the RT primer, which is 2 of the 242nd to 291st base sequences of the prostaglandin E2 receptor gene.
The 48th G is replaced with A to form a new MboI site. SEQ ID NOS: 2 and 3 are the sense side (nucleotide sequences 86 to 115 of the prostaglandin E2 receptor gene) and the antisense side (prostaglandin E2 receptor gene 2 respectively).
64th to 291st base sequence) PCR primer. SEQ ID NO: 4 is the base sequence of the detection probe (the 175th to 209th base sequences of the prostaglandin E2 receptor gene). SEQ ID NO: 5 is
It is the base sequence of the RT primer, and the 656th base is changed from G to A so as to remove the MboI restriction enzyme site present in the 648th to 694th base sequence (original chromosomal DNA) of the mouse β-actin gene. It has been converted. SEQ ID NOS: 6 and 7 are respectively on the sense side (the 519th to 542nd base sequences of the β-actin gene)
And PCR primers for the antisense side (base sequence from 671 to 694 of β-actin gene).

EXAMPLE 1 Kidney nephrons of male ICR mice aged 5 to 7 weeks were prepared from S. Taniguchi et al. (Febbs Letter, 318,
Microdissection according to the method of pp. 65-70, 1993). Transfer the 10 cut glomeruli or 2 mm square renal tubules to a 0.5 ml microtube and add 4M guanidinium thiocyanate, 2
5 mM sodium citrate, 0.5% sarcosyl, 0.1
Denature solution consisting of M2-mercaptoethanol
Add 100 μl of (denaturating solusion) (pH 7.0) and mix well. After adding 5 μg of tRNA,
The nucleic acid is precipitated by adding μl of ethanol, and after centrifugation, the nucleic acid is collected as a pellet. The pellet is washed with 70% ethanol. By this operation, DNA and RNA are simultaneously recovered. The recovered pellet was treated with 10 fmol / 10 μl of RT primer shown in SEQ ID NO: 1 and 50 mM NaC.
1, 4 mM MgCl ₂ , 1 mM DTT, 0.2 mM dN each
10 mM Tris containing TP and 0.01% gelatin (pH
Dissolve in RT buffer consisting of 8.3) and split into two new new tubes. Incubate one tube at 75 ° C for 5 minutes and cool to 42 ° C. 0.5 μl to this
RNase inhibitor (10-50 U / μl) and 0.5 μl of AMV-derived reverse transcriptase (20 U / μl)
Synthesize A.

After reverse transcription, a set of PCR primers shown in SEQ ID NOS: 2 and 3 (200 nM each) and 1.25 U of Taq polymerase were added, and the volume was adjusted to 50 μm with RT buffer.
set to l. 40 cycles of PC heated at denaturing temperature of 94 ° C. for 40 seconds, then at annealing temperature of 60 ° C. for 30 seconds and then at extension temperature of 72 ° C. for 10 seconds
R was done. ₂ μl of PCR product in 10 μl of 12 mM Tris buffer containing 150 mM KCl, 10 mM MgCl 2
MboI (1 u / μl) was added and the mixture was incubated at 37 ° C. for 2 hours. The PCR product treated with MboI was separated by acrylamide gel electrophoresis, and Southern hybridization was carried out with the probe shown in SEQ ID NO: 4 labeled with digoxigenin to detect the PCR product. As a result, a 162 bp band derived from mRNA and a 206 bp band derived from chromosomal DNA were detected (FIGS. 4 and 5).
The outline of this method is shown in FIG.

Example 2 From the mRNA and chromosomal DNA extracted from the tissue obtained in Example 1, cDNA was synthesized in the same manner as in Example 1 using the RT primer shown in SEQ ID NO: 5, and then SEQ ID NO: 6 and PCR was performed in the same manner as in Example 1 using the PCR primers shown in 7. After heating for 40 seconds at a denature temperature of 94 ° C., heating for 30 seconds at an annealing temperature of 68 ° C., and then heating for 10 seconds at an extension temperature of 72 ° C., 40 cycles of PCR were performed. The PCR product was treated with MboI restriction enzyme in the same manner as in Example 1, followed by acrylamide gel electrophoresis and detection and quantification by ethidium bromide staining (FIG. 6).

[0012]

INDUSTRIAL APPLICABILITY The present invention is based on the PCR method and is a further developed method for the quantitative detection of mRNA. As a result, the mRNA can be quantified in an extremely small number of cells or less obtained by biopsy or the like with higher quantification than the conventional method.

[0013]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCAGCGTCGC TGTGACAGGT ACACGAGGAT GACCACCGGG CTGATCAGGA

Sequence number 2: Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGGCCTTTCC CATCACCATG ATGGTCACTG

SEQ ID NO: 3: Sequence Length: 28 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CCAGCGTCGC TGTGACAGGT ACACGAGG

SEQ ID NO: 4: Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGCAAGCGCA AGAAGTCTTT CCTGCTGTGC ATTGG

SEQ ID NO: 5 Sequence length: 47 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TCAGCTGTGG TGGTGAAGCT GTAGCCACTC TCGGTCAGAA TCTTCAT

SEQ ID NO: 6: Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CGTACCACAG GCATTTGTGAT GGA
C

SEQ ID NO: 7 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TCAGCTGTGG TGGTGAAGC TGTA
G

[Brief description of drawings]

FIG. 1 is a schematic diagram of the competitive RT-PCR method of the present invention.

FIG. 2 shows the quantification of PCR with wild-type and mutant DNA.

FIG. 3 shows the quantification of PCR with wild-type and mutant DNA.

FIG. 4 shows detection of prostaglandin E2 receptor mRNA in mouse nephron segment by Southern hybridization by RT-PCR.

FIG. 5 is a graph showing quantification of mRNA expression level by comparison with chromosomal DNA.

FIG. 6 is a diagram showing quantification of the expression level of β-actin mRNA in mouse nephron segment by ethidium bromide staining.

[Explanation of symbols]

 IMCD inner medullary collecting tubule OMCD outer medullary collecting tubule CCD CCD cortical collecting tubule DCT distal curved tubule cTAL cortical ascending thick leg mTAL medullary ascending thick leg DTL descending thick leg PST proximal straight tubular tubing PCT proximal bending urine Capillary glomerulus glomerulus

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shu Kamikobed 3-35-13-509 Sakuradai, Nerima-ku, Tokyo (72) Inventor Kiyoshi Kurokawa 49 Ichigayayanagicho, Shinjuku-ku, Tokyo 401 Ichigaya Hills 401 (72) Inventor Kazuoki Osumi Sanryo Yuka BCL Co., Ltd. 3-30-1 Shimura, Itabashi-ku, Tokyo (72) Inventor Keimi Hata 3-30-1 Shimura Yuka BLC, Itabashi-ku, Tokyo

Claims (2)

[Claims] 1. A cDNA synthesized from an mRNA to be detected, which is derived from a portion of a gene not containing an intron, is c
A method for quantifying mRNA by the RT-PCR method, which comprises amplification with a portion of chromosomal DNA corresponding to DNA. 2. The following steps: 1) For the synthesis of cDNA from an mRNA to be detected derived from a portion not containing an intron of a gene, a new restriction enzyme site not included in the portion to be synthesized is introduced or synthesized. Bases capable of amplifying both the cDNA synthesized in step 1) and the chromosomal DNA of the portion corresponding to the cDNA, using an RT primer selected so as to remove the restriction enzyme site contained in PCR is carried out using a set of PCR primers having a sequence, and PCR containing the RT primer sequence part of step 1)
A product is obtained, 3) the PCR product amplified in step 2) is treated with a restriction enzyme capable of cleaving the site introduced or removed in step 1), and 4) the PCR product treated in step 3) is electrophoresed. PCR product derived from cDNA and PC derived from chromosomal DNA
The method for quantification according to claim 1, which comprises quantifying the mRNA to be detected by separating into R products and 5) quantifying each PCR product.