JPH11155600A - Improvement in determination of expression of cytokine gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately determine the expression of the subject gene by treating a test cell, extracting an mRNA, adding an mRNA which is an external strandard thereto, then synthesizing a cDNA, amplifying and comparing a cytokine DNA and the external standard. SOLUTION: An mRAM as an external standard is added to a test cell population or a treated cell substance or an mRNA extracted therefrom to synthesize a cDNA and the cDNA preparation is then divided to amplify a cytokine DNA from either one of the cDNA preparation. The external standard DNA is amplified from the other DNA preparation to compare both. Thereby, the expression of the objective cytokine gene is accurately determined in a method for crushing or carrying out the lysis treatment of the cell in the test cell population, extracting the mRNA from the treated cell substance, synthesizing the cDNA from the mRNA, amplifying the cDNA and performing the determination when the expression of the specific cytokine gene in a sample cell population is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an improved method for measuring cytokine gene expression, and a kit for performing the method.

[0002]

2. Description of the Related Art As a method for measuring the expression level of a specific cytokine in a cell population, a method using a nucleotide probe that hybridizes to a nucleic acid encoding the cytokine is known, and the sensitivity of this method is improved. For this purpose, it is also possible to synthesize cDNA based on mRNA and amplify it using well-known techniques such as the polymerase chain reaction (PCR), and then measure it.

[0003]

[Related Art] Japanese Patent Application Nos. 9-053528 and 9-2
In the specification of 77580, in the measurement of the expression of a cytokine gene using amplification by PCR, the use of the expression of a gene other than the cytokine gene expressed by a cell population expressing the test cytokine gene as an internal standard is described. It has been described. However, when using this internal standard, for example, when measuring the expression of a cytokine that is increased by stimulating a cell population, the stimulation of the cell population changes the proportion of various cells in the cell population, It is expected that the expression level of the internal standard gene will also change, and it is not suitable as a standard for quantifying the expression level of the cytokine gene.

[0004]

Accordingly, the present invention provides a method using a gene (mRNA) that functions as an external standard.
It is intended to more accurately measure the expression level of a cytokine gene or a change in the expression level of a cytokine gene due to a stimulator.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present invention provides a method for measuring the expression of a specific cytokine gene in a sample cell population by disrupting or lysing cells of the test cell population. Extracting mRNA from the treated cell, synthesizing cDNA from the mRNA,
In the method for amplifying and measuring DNA, the sample cell population or the cell processed product or m extracted therefrom is used.
A cDNA is synthesized from RNA as an external standard,
The present invention provides a method comprising separating a preparation, amplifying cytokine DNA from one cDNA synthesis, amplifying an external standard DNA from the other cDNA preparation, and comparing the two.

[0006] The present invention also provides a method for measuring an increase in the expression level of a specific cytokine gene in a sample cell population by a test stimulant. Prepare an untreated cell population, perform the above measurement on each cell population, and obtain the results obtained from the cell population stimulated with the test stimulant and the results obtained from the cell population not treated with the test stimulant. And comparing the results with the results.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As a sample cell population used in carrying out the method of the present invention, various cell populations such as, for example, tissue of an organism, cultured cells of an organism and the like are used. First, mRNA is extracted from the sample cell population according to a conventional method, and then cDNA is synthesized based on the mRNA according to a conventional method, and the cDNA is amplified using specific primers.
Detect and measure NA. A probe can be used for detection and measurement of cDNA. In this method, mRNA as an external standard is added to the sample cell population obtained as described above or mRNA extracted from the cell population, and cDNA synthesis, amplification and detection are performed. In this case, for amplification, detection and measurement, a primer pair and a probe specific to a cytokine gene and a primer pair and a probe specific to an external control mRNA may be used separately. The primer pair and the probe will be described later in detail.

[0008] To measure a change, eg, an increase or decrease, in the expression of a cytokine gene when a cell population is stimulated with a specific test substance, a cell population that has not been stimulated with the test substance and a test substance that is more suitable for the purpose are used. A cell population stimulated under desired conditions is prepared, the expression of a specific cytokine gene is measured for each cell population by the above-described method, and the results may be compared. The rate of increase (SI) in the expression of a specific cytokine gene by stimulation with a specific test substance can be calculated by the following formula.

[0009]

(Equation 1) The external control mRNA is not particularly limited, and for example, mRNA of rat or mouse glyceraldehyde-2-phosphate dehydrogenase (GAPDH) gene can be used.

The cytokines encoded by the gene to be measured according to the present invention include, for example, those associated with allergy as follows. IL-2: acts as a T cell growth factor and induces the proliferation of Th1 cells, which are thought to play an important role in delayed allergy. It is thought that the inducibility can be evaluated. IL-10: has a function of controlling the proliferation of Th1-type cells, and the index indicates that the inhibition of the proliferation of Th1-type cells is released by changing the cytokine in a negative direction. It may be useful for allergic evaluation.

IL-12: IL-12 is p40 and p35
A heterodimer consisting of two subunits, Th0
Induces differentiation of cells into Th1 cells. Therefore, T
It is considered that evaluating the induction of h1 cells to cells is useful for evaluating allergic properties. IFN-γ: a cytokine produced from Th1 cells, it is considered that by evaluating this, activation of Th1 cells can be evaluated, which is useful for allergic evaluation.

Next, the method for amplifying and detecting cDNA in the method of the present invention will be described. Polymerase chain reaction (PCR) is widely used as a nucleic acid amplification method. As one application of the PCR method, there is a method of measuring a nucleic acid using a probe in which a reporter dye 1 and a quencher dye 2 are bound. In this method, the reporter dye 1 hybridizes to a site on the template sandwiched between a site on the template to which the forward primer used in the PCR method hybridizes and a site on the template to which the reverse primer hybridizes. And a quencher dye 2 bound probe,
A PCR reaction is performed using a DNA polymerase having 5 ′ → 3 ′ exonuclease activity.

For example, in FIGS.
When the forward primer is extended by the use of the DNA polymerase to the probe, the oligonucleotide constituting the probe is degraded by the action of the 5 ′ → 3 ′ exonuclease activity of the DNA polymerase, and the extension of the forward primer is subsequently performed. A product forms.

In this case, the reporter 1 and the quencher 2
While the is bound to the probe, it does not emit fluorescence due to the interaction between the two, but if the oligonucleotide constituting the probe is degraded with the extension of the primer, the reporter 1 and the quencher 2 are separated, and the reporter 1 emits fluorescence when irradiated with ultraviolet or argon laser because it is not affected by the quencher 2. Therefore, by selecting primers and probes that specifically hybridize to a specific DNA, a specific nucleic acid can be selectively measured based on the fluorescence intensity.

[0015] This method has already been described in Taq ManPCR.
(Trademark) etc. In the above method, it is necessary to select a pair of probes having the above-described positional relationship on the test nucleic acid, that is, the template nucleic acid, and the probe, but not only the primer but also the primer under the same hybridization conditions. The probe must hybridize to the template nucleic acid early. Because
If the extension product of the primer (single-stranded nucleic acid) extends beyond the position where the probe should hybridize, the probe can no longer hybridize, and therefore the 5 'to 3' exonuclease of DNA polymerase It cannot be decomposed by the activity.

When two nucleic acids whose specific nucleotide sequences are known hybridize, the likelihood of hybridization can be estimated to some extent by calculation of the melting point (Tm). However, the combination of the primer and probe selected by this estimation is not always
It does not give good results in the NA measurement method, and it is necessary to select a probe and primer combination by trial and error for a specific nucleic acid to be measured.

Rat GA is an example of an external control mRNA.
When using the mRNA of the PDH gene, for example, from position 111 to SEQ ID NO: 1 as a forward primer
Using an oligonucleotide that hybridizes to the region at position 136, an oligonucleotide that hybridizes to the region from position 266 to position 284 in SEQ ID NO: 1 as a reverse primer, and SEQ ID NO: as a probe
Oligonucleotides that hybridize to the region at positions 234 to 258 in 1 can be used.

As a specific example, a DNA using the nucleotide sequence of 5'-GTTACCAGGGCTGCCTTCTC-3 '(SEQ ID NO: 3) is used as a forward primer, and 5'-GGGTTTCCCGTTGATGACC-3' is used as a reverse primer.
DNA using the base sequence of (SEQ ID NO: 4) was used, and 5′-AACGGCACAGTCAAGGCTGAGAAT was used as a probe.
DNA having the sequence of G-3 ′ (SEQ ID NO: 5), or substitution of four or less bases in the base sequence thereof,
DNA modified by deletion and / or addition and capable of hybridizing to the corresponding region is used as a primer and a probe.

As another example of the external control mRNA, when using mouse GAPDH gene mRNA, positions 92 to 111 in SEQ ID NO: 2 can be used as forward primers.
241 of SEQ ID NO: 2 was used as a reverse primer using an oligonucleotide that hybridized to the
Oligonucleotides that hybridize to the region from position 259 to position 259 and hybridize to the region from position 209 to position 234 in SEQ ID NO: 2 as a probe; or a region from nucleotides 46 to 67 as one primer Oligonucleotides that hybridize to the region of nucleotides 252 to 271 can be used as the other primer, and oligonucleotides in the region of nucleotides 222 to 242 can be used as probes.

As a specific example, a DNA using the sequence of GTCACCAGGGCTGCCATTTG (SEQ ID NO: 6) is used as a forward primer, and TG is used as a reverse primer.
DNA having the nucleotide sequence of TCATCAACGGGAAGCCC (SEQ ID NO: 7) was used, and AACCGGCACAGTCAA was used as a probe.
DN having the nucleotide sequence of GGCCGAGAATGG (SEQ ID NO: 8)
A; or 5'AATG as a forward primer
Using a DNA having the nucleotide sequence of GTGAAGGTCGGTGTGAAC3 '(SEQ ID NO: 41), 5'
A DNA primer having the base sequence of GAAGATGGTGATGGGCTTCC3 '(SEQ ID NO: 42) was used, and 5' CAAGCTTCCCATTCTCGGCC3 '(SEQ ID NO: 43) was used as a probe.
Using a DNA having the nucleotide sequence of
In A, primers and probes modified by substitution, deletion and / or addition of 4 or less bases and capable of hybridizing to the corresponding region can be used.

In order to amplify and measure mRNA or cDNA of a gene encoding a cytokine, for example, the following primer pair and probe can be used. (1) In order to measure mRNA or cDNA encoding interleukin-2, in FIG.
An oligonucleotide that hybridizes to the region of nucleotides 202 to 226 of the nucleic acid encoding interleukin-2 is used as the one primer, and nucleotides 345 to 369 of the nucleic acid are used as the other primer.
And an oligonucleotide that hybridizes to the region of nucleotides 242 to 268 in the nucleic acid as the probe; or (b) encoding interleukin-2 as the one primer. An oligonucleotide that hybridizes to the region of nucleotides 22 to 43 of the nucleic acid to be used, an oligonucleotide that hybridizes to the region of nucleotides 83 to 105 of the nucleic acid as the other primer, and nucleotide 49 of the nucleic acid as the probe. An oligonucleotide that hybridizes to the region of ~ 74 is used.

FIG. 4A shows the positional relationship between the primer binding region and the probe binding region on the gene encoding interleukin-2 (IL-2). In this figure, the underlined portion upstream is the region to which the forward primer hybridizes, the underlined portion downstream is the region to which the reverse primer hybridizes, and the underlined portion between them is the region to which the probe hybridizes. It is.

The nucleotide sequences (a) of preferred primers and probes for measuring the interleukin-2 (IL-2) gene are as follows (indicated by one underline in (A) of FIG. 4). Forward primer 5'TGATGGACCTACAGGAGCTCCT
GAG3 '(SEQ ID NO: 14) Reverse primer 5' GAGTCAAATCCAGAACATGCCGCAG
3 '(SEQ ID NO: 15) Probe 5' CAGGAACCTGAAACTCCCCAGGATGCT3 '(SEQ ID NO: 16)

Other nucleotide sequences (b) of preferred primers and probes for measuring interleukin-2 (IL-2) gene are as follows (2 in FIG. 4 (A)).
This is underlined). Forward primer 5'TCACAGTGACCTCAAGTCCTGC
3 '(SEQ ID NO: 29) Reverse primer 5' TGTTGACAAGGAGCACAAGTGTC3 '
(SEQ ID NO: 30) Probe 5 'TGTACAGCATGCAGCTCGCATCCTGT 3' (SEQ ID NO: 31)

(2) In order to measure mRNA or cDNA encoding interferon-γ, in FIG.
Using an oligonucleotide that hybridizes to the region of nucleotides 430 to 450 in the nucleic acid encoding
An oligonucleotide that hybridizes to the region of ５６564 is used, and an oligonucleotide that hybridizes to the region of nucleotides 508 to 530 in the nucleic acid is used as the probe.

FIG. 4B shows the positional relationship between the primer binding region and the probe binding region on the gene encoding interferon-γ. In this figure, the meaning of the three underlines is as described for FIG. Preferred nucleotide sequences of primers and probes for measuring the interferon-γ gene are as follows. Forward primer 5'GTCAACAACCCACAGGTCCAG
3 '(SEQ ID NO: 17) Reverse primer 5'TTGGGACAATCTCTTCCCCA3'
(SEQ ID NO: 18) Probe 5'CGACTCCTTTTCCGCTTCCTGAG3 '(SEQ ID NO: 19)

(3) In order to measure mRNA or cDNA encoding interleukin-10, in FIG. 5, (a) nucleotides 381-4 in the nucleic acid encoding interleukin-10 were used as the one primer.
02, an oligonucleotide that hybridizes to the region of nucleotides 630 to 650 in the nucleic acid as the other primer, and an oligonucleotide that hybridizes to the region of nucleotides 431 to 453 in the nucleic acid as the probe. Alternatively, an oligonucleotide that hybridizes is used; or (b) an oligonucleotide that hybridizes to the region of nucleotides 15 to 38 in the nucleic acid encoding interleukin-10 is used as the one primer, and the oligonucleotide is used as the other primer. Using an oligonucleotide that hybridizes to the region of nucleotides 90 to 112,
And nucleotide 6 in the nucleic acid as the probe
Oligonucleotides that hybridize to regions 2-87 are used.

FIG. 5C shows the positional relationship between the primer binding region and the probe binding region on the gene encoding interleukin-10 (IL-10). In this figure, the meaning of the three underlines is as described for FIG. The nucleotide sequences (a) of preferred primers and probes for measuring the interleukin-10 gene are as follows. Forward primer 5'CCCAGAAATCAAGGAGCATTTG
3 '(SEQ ID NO: 20) Reverse primer 5' CCTGGAGTCCAGCAGACTCAA3 '
(SEQ ID NO: 21) Probe 5 'TCAGGATGCGGCTGAGGCGCTGT3' (SEQ ID NO: 22) Other nucleotide sequences (b) of preferred primers and probes for measuring the interleukin-10 gene are as follows. Forward primer 5'AGAGACTTGCTCTTGCACTACC
AA3 '(SEQ ID NO: 32) Reverse primer 5' GTAAGAGCAGGCAGCATAGCAGT3 '
(SEQ ID NO: 33) Probe 5'TGAGCCAGGCATGATGGAGCTCTCTT3 '(SEQ ID NO: 34)

(4) In order to measure mRNA or cDNA encoding the interleukin-12p35 subunit, FIG. 6 shows (a) nucleotide 571 in the nucleic acid encoding the interleukin-12p35 subunit as the one primer. Oligonucleotides that hybridize to the region 594, and nucleotides 693-71 in the nucleic acid as the other primer.
An oligonucleotide that hybridizes to the region of nucleotides 657 to 680 in the nucleic acid as the probe; or (b) an interleukin-12p35 subunit as the one primer. Using an oligonucleotide that hybridizes to the region of nucleotides 62 to 81 in the nucleic acid encoding the nucleotides 208 to 22 in the nucleic acid as the other primer
An oligonucleotide that hybridizes to the region of nucleotide No. 9 is used, and an oligonucleotide that hybridizes to the region of nucleotides 118 to 143 in the nucleic acid is used as the probe.

FIG. 6D shows the positional relationship between the primer binding site and the probe binding site on the gene encoding the interleukin-12p35 subunit. In this figure, the meaning of the three underlines is as described for FIG. The nucleotide sequences (a) of preferred primers and probes for measuring the interleukin-12p35 subunit gene are as follows. Forward primer 5'ATCATTCTAGACAAGGGCATGC
TG3 '(SEQ ID NO: 23) Reverse primer 5'GTGAAGCAGGATGCAGAGCTTC3'
(SEQ ID NO: 24) Probe 5 'TAAGGGTCTGCTTCTGCTTCTCCCACAGGA3'
(SEQ ID NO: 25)

Other nucleotide sequences (b) of preferred primers and probes for measuring the interleukin-12p35 subunit gene are as follows. Forward primer 5'CCAAGGTCAGCGTTCCAACA3
′ (SEQ ID NO: 35) Reverse primer 5 ′ TAAGACACCTGGCAGGTCCAGA3 ′
(SEQ ID NO: 36) Probe 5'CGGTCCAGCATGTGTCAATCACGCTA3 '(SEQ ID NO: 37)

(5) In order to measure mRNA or cDNA encoding the interleukin-12 p40 subunit, FIG. 7 shows (a) nucleotide 600 in the nucleic acid encoding the interleukin-12 p40 subunit as the one primer. Oligonucleotides that hybridize to the region from 639 to 619, and nucleotides 739 to 76
An oligonucleotide that hybridizes to the region of nucleotides 701 to 726 in the nucleic acid as the probe; or (b) an interleukin-12p40 subunit as the one primer. An oligonucleotide that hybridizes to the region of nucleotides 199 to 221 in the nucleic acid encoding the unit is used, and nucleotide 430 in the nucleic acid is used as the other primer.
An oligonucleotide that hybridizes to the region of ４449 is used, and an oligonucleotide that hybridizes to the region of nucleotides 333 to 358 in the nucleic acid is used as the probe.

FIG. 7E shows the positional relationship between the primer binding site and the probe binding site on the gene encoding the interleukin-12p40 subunit. In this figure, the meaning of three underlines is as described in FIG. The nucleotide sequences (a) of preferred primers and probes for measuring the interleukin-12p40 subunit gene are as follows. Forward primer 2 5'TGTCCTGCCAGGAGGATGTC
3 '(SEQ ID NO: 26) Reverse primer 5' CTTCATCTGCAAGTTCTTGGGC3 '
(SEQ ID NO: 27) Probe 5'ATGATGTCCCTGATGAAGAAGCTGGT3 '(SEQ ID NO: 28) Other nucleotide sequences (b) of preferred primers and probes for measuring the interleukin-12p40 subunit gene are as follows. Forward primer 2 5'AGATGACATCACCTGGACCT
CAG3 '(SEQ ID NO: 38) Reverse primer 5' ACGTGAACCGTCCGGAGTAA3 '
(SEQ ID NO: 39) Probe 5'TTCCTTCTTGTGGAGCAGCAGATGTG3 '(SEQ ID NO: 40)

The oligonucleotides for the above various primers and probes have 15 to 35 nucleotides,
And it is preferably 20 to 30 pieces. This is because if the size of the primer and the probe is long, it is difficult to hybridize to the single-stranded DNA, and if the size is too short, the specificity of hybridization is reduced.

The above specific nucleotide sequences of primers and probes are particularly preferred sequences,
It is known that a primer or probe consisting of 0 nucleotides hybridizes even if a small number of mismatches exist with the template strand, and can function as a primer for PCR or as a detection probe. Therefore, the primers and probes of the present invention are not limited to those having the above specific nucleotide sequence, and may be modified by substitution, deletion and / or addition of 4 or less nucleotides to the above specific nucleotide sequence. Primers and probes that are capable of hybridizing to a predetermined region are also included in the present invention.

The probe used in the present invention has a reporter dye attached to one end, eg, the 5′-end, and a quencher dye attached to the other end, eg, the 3′-end. Whereas a reporter dye is a substance that emits fluorescence upon irradiation with, for example, ultraviolet light or an argon laser, a quencher acts on the reporter dye when present in close proximity to the reporter dye to eliminate the generation of fluorescence. It has the action of doing. As a reporter dye, for example, 6-carboxy-fluorescein (F
AM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE), hexochloro-6-carboxyfluorescein (HEX), etc. On the other hand, as a quencher dye, 6-carboxy-tetramethyl-rhodamine (TAMRA) or the like is used.

The binding of the reporter dye and the quencher dye to the probe oligonucleotide is performed, for example, by using a few methylene chains as a linker on the 5 'side of the probe and adding a FAM molecule to the terminal phosphate group in the form of a phosphate ester. And on the 3 ′ side via a structural unit shown below,
The TAMRA molecule is linked by an amide bond.

[0038]

Embedded image

The present invention further provides a kit for use in performing the above-described method. This kit comprises the primers and probes defined above.

[0040]

Next, the present invention will be described more specifically with reference to examples. General Methods Isolation of RNA from Test Sample Cell Populations are placed in 2.2 ml tubes and centrifuged at 2000 × g for 5 minutes at 4 ° C. to remove the culture. 1 ml of ISOGEN (Nippon Gene) is added to the remaining cells, stirred, and left at room temperature for 5 minutes. Add 0.2 ml of chloroform,
Stir for 15 seconds. 120 minutes after standing at room temperature for 2-3 minutes
After centrifugation at 00 × g for 15 minutes at 4 ° C., transfer the aqueous layer to another tube. 0.5 ml of isopropanol was added thereto and 5
After leaving at room temperature for -10 minutes, 12,000 Xg, 10 minutes, 4
Centrifuge at ℃ to obtain a precipitate. 75 in the resulting precipitate
% Ethanol is added and centrifuged at 12,000 × g for 5 minutes at 4 ° C. to obtain a precipitate. The precipitate is air-dried and then dissolved in distilled water (this solution is referred to as an RNA solution).

Preparation of mRNA as External Standard Same as the method for isolating RNA from the test sample cell population, except that after adding ISOGEN to the cells,
1 of 0.5 μg / ml rat poly (A) ⁺ RNA
(TOYOBO) is added.

Synthesis of cDNA 5 × F was added to 10.5 μl of an RNA solution containing 1 μl of RNA.
first Strand Buffer (Gibco
BRL) 4 μl, 0.1 M DTT (Gibco BR
L) 2 μl, 0.5 mg / ml oligo (dT) _12-18
1 μl of Primer (Gibco BRL), 2.5 mM
dNTP (dATP, dTTP, dGTP, dCT
P) (Takara Shuzo) 1 μl, 124 U / μl RNase
Inhibitor (Takara Shuzo) 0.5 μl, 200 U /
μl M-MLV Reverse Transcript
After leaving 1 μl of the mixture (case (Gibco BRL)) at room temperature for 10 minutes, the mixture is incubated at 37 ° C. for 50 minutes.

Measurement of cDNA 5 μl of cDNA, 10 × PCR buffer (Perkin
Elmer) 5 μl, 25 mM MgCl ₂ (Perk)
in Elmer) 7 μl, 20 μM forward primer 0.75 μl, 20 μM reverse primer 0.
75 μl, 3 μM probe 5 μl, 2.5 mM dATP
(Perkin Elmer) 1 μl, 2.5 mM dG
1 μl of TP (Perkin Elmer), 2.5 mM
1 μl of dCTP (Perkin Elmer), 5 mM
dUTP (Perkin Elmer) 1 μl, distilled water 21.75 μl, AmpErase ^R UNG (Perk
in Elmer) 0.5 μl, AmpliTaq ^R D
NA Polymerase (Perkin Elme
r) 0.25 μl of the mixture was added to ABI PRISM770
0 (Perkin Elmer), and perform a PCR reaction. Temperature conditions are 50 ° C for 2 minutes and 95 ° C for 10 minutes.
After keeping the temperature for 5 minutes, (at 95 ° C. for 15 seconds, 64.5 ° C.
For 1 minute) for 40 times, and the fluorescence intensity is measured for each cycle.

Embodiment 1 Encoding rat GAPDH
Standard curve of mRNA of gene of interest The initial molecular weight (concentration) of the gene (template) encoding rat GAPDH and the number of PCR cycles (Thresh) until the fluorescence intensity departs from the baseline and starts to increase
old Cycle) were tested the relationship between the C _T. The template gene, forward primer, reverse primer and probe were as follows.

Template: Rat GAPDH cDNA (Nu
cleic Acids Res. 13 (5), 143
1-1144 (1985)) Forward primer: SEQ ID NO: 3 Reverse primer: SEQ ID NO: 4 Probe: SEQ ID NO: 5 Reporter dye: FAM Quencher dye: TAMRA Logarithm of initial template molecule number of cDNA of rat GAPDH and Ct FIG. 8 shows an example of a standard curve showing the relationship with.

The initial template molecular weight (concentration) and Ct are well correlated, and it is clear that rat GAPDH is useful as an external standard. FIG. 9 shows the results when the same amount (5 mg) of rat poly (A) ⁺ RNA was added to the same sample (cell population) (n = 3). It is clear that the reproducibility is very high and the measurement can be performed with high accuracy. Rat poly (A) ⁺ RN added to mouse RNA sample
FIG. 10 shows the relationship between the amount of A and the amount (copy number) of the amplified rat GAPDH cDNA. Poly (A) ⁺ R
The amount of NA and the amount of generated cDNA are well proportional. From these results, it was found that poly (A) ⁺ RN as an external standard mRNA
It turns out that 5 ng of A is sufficient.

Embodiment 2 FIG . Encode each cytokine
Standard curve for gene measurement Initial molecular weight (concentration) of the gene (template) encoding each cytokine, and the number of PCR cycles (Thresho) until the fluorescence intensity departs from the baseline and starts to increase.
It was tested the relationship between the ld Cycle) C _T. The template gene, forward primer, reverse primer and probe were as follows.

Interleukin-2 gene (a) Template: mouse interleukin-2 (Nature 31)
3 (600) 402-404 (1985)) Forward primer: SEQ ID NO: 14 Reverse primer: SEQ ID NO: 15 Probe: SEQ ID NO: 16 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-2 gene (b) template: mouse interleukin-2 (Nature 31)
3 (600) 402-404 (1985)) Forward primer: SEQ ID NO: 29 Reverse primer: SEQ ID NO: 30 Probe: SEQ ID NO: 31 Reporter dye: FAM Quencher dye: TAMRA

Interferon gene template: mouse interferon-γ (Proc. Natl. Aca
d. Sic. USA 80, 5842-5846 (198
3)) Forward primer: SEQ ID NO: 17 Reverse primer: SEQ ID NO: 18 Probe: SEQ ID NO: 19 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-10 gene (a) template: mouse interleukin-10 (Science)
248: 1230-1234 (1990)) Forward primer: SEQ ID NO: 20 Reverse primer: SEQ ID NO: 21 Probe: SEQ ID NO: 22 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-10 gene (b) template: mouse interleukin-10 (Science)
248: 1230-1234 (1990)) Forward primer: SEQ ID NO: 32 Reverse primer: SEQ ID NO: 33 Probe: SEQ ID NO: 34 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-12p35 subunit
(A) Template: mouse interleukin-12 (J. Immun
ol. 148, 3433-3440 (1992)) Forward primer: SEQ ID NO: 23 Reverse primer: SEQ ID NO: 24 Probe: SEQ ID NO: 25 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-12p35 subunit
(B) Template: mouse interleukin-12 (J. Immun
ol. 148, 3433-3440 (1992)) Forward primer: SEQ ID NO: 35 Reverse primer: SEQ ID NO: 36 Probe: SEQ ID NO: 37 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-12p40 subunit
Gene (a) template: mouse interleukin-12 (J. Immun
ol. 148, 3433-3440 (1992)) Forward primer: SEQ ID NO: 26 Reverse primer: SEQ ID NO: 27 Probe: SEQ ID NO: 28 Reporter dye: FAM Quencher dye: TAMRA

Interleukin-12p40 subunit
Gene (b) template: mouse interleukin-12 (J. Immun
ol. 148, 3433-3440 (1992)) Forward primer: SEQ ID NO: 38 Reverse primer: SEQ ID NO: 39 Probe: SEQ ID NO: 40 Reporter dye: FAM quencher dye: TAMRA The results are shown in FIGS. These results, there is a linear relationship between the C _T number of molecules of the nucleic acid (concentration) is also in the measurement of either gene, it was found to be capable of accurate measurement of nucleic acid by the method of the present invention.

[0057]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 300 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA Sequence CTCTCTGCTC CTCCCTGTTC TAGAGACAGC CGCATCTTCT TGTGCAGTGC CAGCCTCGTC 60 TCATAGACAA GATGGTGAAG GTCGGTGTGA ACGGATTTGG CCGTATCGTCCGCGCTCGTCCCTGCGCCGTATGCTC ACATTGTTGC CATCAACGAC CCCTTCATTG 180 ACCTCAACTA CATGGTCTAC ATGTTCCAGT ATGACTCTAC CCACGGCAAG TTCAACGGCA 240 CAGTCAAGGC TGAGAATGGG AAGCTGGTCA TCAACGGGAA ACCCATCACC ATCTTCCAGG 300

SEQ ID NO: 2 Sequence length: 300 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ACAGCCGCAT CTTCTTGTGC AGTGCCAGCC TCGTCCCGTA GACAAAATGG TGAAGGTCGG 60 TGTGAACGGA TTTGGCCGTA TTGGGCGCGC GGTCCATCGCGGCT AGTGGAGATT GTTGCCATCA ACGACCCCTT CATTGACCTC AACTACATGG TCTACATGTT 180 CCAGTATGAC TCCACTCACG GCAAATTCAA CGGCACAGTC AAGGCCGAGA ATGGGAAGCT 240 TGTCATCAAC GGGAAGCCCA TCACCATCTT CCAGGAGCGA GACCCCACTA ACATCAAATG 300

SEQ ID NO: 3 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GTTACCAGGG CTGCCTTCTC 20

SEQ ID NO: 4 Sequence length: 19 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGGTTTCCCG TTGATGACC 19

SEQ ID NO: 5 Sequence length: 25 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence AACGGCACAG TCAAGGCTGA GAATG 25

SEQ ID NO: 6 Sequence length: 20 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: Synthetic DNA sequence GTCACCAGGG CTGCCATTTG 20

SEQ ID NO: 7 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TGTCATCAAC GGGAAGCCC 19

SEQ ID NO: 8 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence AACGGCACAG TCAAGGCCGA GAATGG 26

SEQ ID NO: 9 Sequence length: 420 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATCACCCTTG CTAATCACTC CTCACAGTGA CCTCAAGTCC TGCAGGCATG TACAGCATGC 60 AGCTCGCATC CTGTGTCACA TTGACACTTG TGCTCCTTCCACT GCTCCACTTC AAGCTCTACA GCGGAAGCAC AGCAGCAGCA GCAGCAGCAG CAGCAGCAGC 180 AGCAGCACCT GGAGCAGCTG TTGATGGACC TACAGGAGCT CCTGAGCAGG ATGGAGAATT 240 ACAGGAACCT GAAACTCCCC AGGATGCTCA CCTTCAAATT TTACTTGCCC AAGCAGGCCA 300 CAGAATTGAA AGATCTTCAG TGCCTAGAAG ATGAACTTGG ACCTCTGCGG CATGTTCTGG 360 ATTTGACTCA AAGCAAAAGC TTTCAATTGG AAGATGCTGA GAATTTCATC AGCAATATCA 420

SEQ ID NO: 10 Sequence length: 180 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence AAGTTTGAGG TCAACAACCC ACAGGTCCAG CGCCAAGCAT TCAATGAGCT CATCCGAGTG 60 GTCCACCAGC TGTTGCCGGA ATCCAGCCTC AGGAAGCGGATCGATCGACGATC GGGGTGGGGA AGAGATTGTC CCAATAAGAA TAATTCTGCC AGCACTATTT GAATTTTTAA 180

SEQ ID NO: 11 Sequence length: 660 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA sequence GGGGGGGGGG ATTTAGAGAC TTGCTCTTGC ACTACCAAAG CCACAAAGCA GCCTTGCAGA 60 AAAGAGAGCT CCATCATGCC TGGCTCCTGCTCACTGCTACTCT ATGAGGATCA GCAGGGGCCA GTACAGCCGG GAAGACAATA ACTGCACCCA CTTCCCAGTC 180 GGCCAGAGCC ACATGCTCCT AGAGCTGCGG ACTGCCTTCA GCCAGGTGAA GACTTTCTTT 240 CAAACAAAGG ACCAGCTGGA CAACATACTG CTAACCGACT CCTTAATGCA GGACTTTAAG 300 GGTTACTTGG GTTGCCAAGC CTTATCGGAA ATGATCCAGT TTTACCTGGT AGAAGTGATG 360 CCCCAGGCAG AGAAGCATGG CCCAGAAATC AAGGAGCATT TGAATTCCCT GGGTGAGAAG 420 CTGAAGACCC TCAGGATGCG GCTGAGGCGC TGTCATCGAT TTCTCCCCTG TGAAAATAAG 480 AGCAAGGCAG TGGAGCAGGT GAAGAGTGAT TTTAATAAGC TCCAAGACCA AGGTGTCTAC 540 AAGGCCATGA ATGAATTTGA CATCTTCATC AACTGCATAG AAGCATACAT GATGATCAAA 600 ATGAAAAGCT AAAACACCTG CAGTGTGTAT TGAGTCTGCT GGACTCCAGG ACCTAGACAG 660

SEQ ID NO: 12 Sequence length: 660 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Sequence CCCAAGGTCA GCGTTCCAAC AGCCTCACCC TCGGCATCCA GCAGCTCCTC TCAGTGCCGG 60 TCCAGCATGT GTCAATCACG CTACCTCCCATCTGTTTTTGCCCACCTG CTCAGTTTGG CCAGGGTCAT TCCAGTCTCT GGACCTGCCA GGTGTCTTAG CCAGTCCCGA 180 AACCTGCTGA AGACCACAGA TGACATGGTG AAGACGGCCA GAGAAAAACT GAAACATTAT 240 TCCTGCACTG CTGAAGACAT CGATCATGAA GACATCACAC GGGACCAAAC CAGCACATTG 300 AAGACCTGTT TACCACTGGA ACTACACAAG AACGAGAGTT GCCTGGCTAC TAGAGAGACT 360 TCTTCCACAA CAAGAGGGAG CTGCCTGCCC CCACAGAAGA CGTCTTTGAT GATGACCCTG 420 TGCCTTGGTA GCATCTATGA GGACTTGAAG ATGTACCAGA CAGAGTTCCA GGCCATCAAC 480 GCAGCACTTC AGAATCACAA CCATCAGCAG ATCATTCTAG ACAAGGGCAT GCTGGTGGCC 540 ATCGATGAGC TGATGCAGTC TCTGAATCAT AATGGCGAGA CTCTGCGCCA GAAACCTCCT 600 GTGGGAGAAG CAGACCCTTA CAGAGTGAAA ATGAAGCTCT GCATCCTGCT TCACGCCTTC 660

SEQ ID NO: 13 Sequence length: 600 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence CTGTGACACG CCTGAAGAAG ATGACATCAC CTGGACCTCA GACCAGAGAC ATGGAGTCAT 60 AGGCTCTGGA AAGACCCTGA CCATCACTGT CAAAGAGTTT CTAGATCG 120 CTGCCACAAA GGAGGCGAGA CTCTGAGCCA CTCACATCTG CTGCTCCACA AGAAGGAAAA 180 TGGAATTTGG TCCACTGAAA TTTTAAAAAA TTTCAAAAAC AAGACTTTCC TGAAGTGTGA 240 AGCACCAAAT TACTCCGGAC GGTTCACGTG CTCATGGCTG GTGCAAAGAA ACATGGACTT 300 GAAGTTCAAC ATCAAGAGCA GTAGCAGTTC CCCTGACTCT CGGGCAGTGA CATGTGGAAT 360 GGCGTCTCTG TCTGCAGAGA AGGTCACACT GGACCAAAGG GACTATGAGA AGTATTCAGT 420 GTCCTGCCAG GAGGATGTCA CCTGCCCAAC TGCCGAGGAG ACCCTGCCCA TTGAACTGGC 480 GTTGGAAGCA CGGCAGCAGA ATAAATATGA GAACTACAGC ACCAGCTTCT TCATCAGGGA 540 CATCATCAAA CCAGACCCGC CCAAGAACTT GCAGATGAAG CCTTTGAAGA ACTCACAGGT 600

SEQ ID NO: 14 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TGATGGACCT ACAGGAGCTC CTGAG 25

SEQ ID NO: 15 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence GAGTCAAATC CAGAACATGC CGCAG 25

SEQ ID NO: 16 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CAGGAACCTG AAACTCCCCA GGATGCT 27

SEQ ID NO: 17 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence GTCAACAACC CACAGGTCCA G 21

SEQ ID NO: 18 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TTGGGACAAT CTCTTCCCCA 20

SEQ ID NO: 19 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CGACTCCTTT TCCGCTTCCT GAG 23

SEQ ID NO: 20 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CCCAGAAATC AAGGAGCATT TG 22

SEQ ID NO: 21 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CCTGGAGTCC AGCAGACTCA A 21

SEQ ID NO: 22 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TCAGGATGCG GCTGAGGCGC TGT 23

SEQ ID NO: 23 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence ATCATTCTAG ACAAGGGCAT GCTG 24

SEQ ID NO: 24 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence GTGAAGCAGG ATGCAGAGCT TC 22

SEQ ID NO: 25 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TAAGGGTCTG CTTCTGCTTC TCCCACAGGA 30

SEQ ID NO: 26 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TGTCCTGCCA GGAGGATGTC 20

SEQ ID NO: 27 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CTTCATCTGC AAGTTCTTGG GC 22

SEQ ID NO: 28 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence ATGATGTCCC TGATGAAGAA GCTGGT 26

SEQ ID NO: 29 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TCACAGTGAC CTCAAGTCCT GC 22

SEQ ID NO: 30 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TGTTGACAAG GAGCACAAGT GTC 23

SEQ ID NO: 31 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TGTACAGCAT GCAGCTCGCA TCCTGT 26

SEQ ID NO: 32 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence AGAGACTTGC TCTTGCACTA CCAA 24

SEQ ID NO: 33 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence GTAAGAGCAG GCAGCATAGC AGT 23

SEQ ID NO: 34 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TGAGCCAGGC ATGATGGAGC TCTCTT 26

SEQ ID NO: 35 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CCAAGGTCAG CGTTCCAACA 20

SEQ ID NO: 36 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TAAGACACCT GGCAGGTCCA GA 22

SEQ ID NO: 37 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CGGTCCAGCA TGTGTCAATC ACGCTA 26

SEQ ID NO: 38 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence AGATGACATC ACCTGGACCT CAG 23

SEQ ID NO: 39 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence ACGTGAACCG TCCGGAGTAA 20

SEQ ID NO: 40 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence TTCCTTCTTG TGGAGCAGCA GATGTG 26

SEQ ID NO: 41 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence AATGGTGAAG GTCGGTGTGA AC 22

SEQ ID NO: 42 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence GAAGATGGTG ATGGGCTTCC 20

SEQ ID NO: 43 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Synthetic DNA sequence CAAGCTTCCC ATTCTCGGCC 20

[Brief description of the drawings]

FIG. 1 is a diagram showing a part of the nucleotide sequence of a gene encoding rat glyceraldehyde-3-phosphate dehydrogenase, and the positions of primers and probes corresponding thereto.

FIG. 2 is a diagram showing a part of the nucleotide sequence of a gene encoding mouse glyceraldehyde-3-phosphate dehydrogenase and the positions of primers and probes corresponding thereto.

FIG. 3 is a diagram showing the principle of the measurement method of the present invention.

FIG. 4 is a diagram showing a part of the nucleotide sequence of a gene encoding interleukin-2 (A) and interferon-γ (B), and the positions of primers and probes corresponding thereto.

FIG. 5 is a diagram showing a part of the nucleotide sequence of a gene encoding interleukin-10 (C) and the positions of primers and probes relative thereto.

FIG. 6 is a diagram showing a part of the nucleotide sequence of the gene encoding the interleukin-12p35 subunit (D) and the positions of primers and probes relative thereto.

FIG. 7 is a diagram showing a part of the nucleotide sequence of the gene encoding interleukin-12p40 subunit (E) and the positions of primers and probes corresponding thereto.

Figure 8 is a graph showing as measured by the method of the present invention to the gene encoding rat-glyceraldehyde-3-phosphate dehydrogenase, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid It is.

FIG. 9 shows the same amount (5 ng) of rat p in the same sample.
It is a graph which shows the agreement of the measured value at the time of amplifying a glyceraldehyde-3-phosphate dehydrogenase gene by adding poly (A) ⁺ RNA.

FIG. 10 shows that various amounts of rat poly were added to the sample.
(A) A graph showing that the amount of cDNA generated when a glyceraldehyde-3-phosphate dehydrogenase gene is amplified by adding ⁺ RNA is proportional to the amount of added RNA.

Figure 11, as measured by the method of the present invention the gene coding for the interleukin-2 is a graph showing the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 12 is a graph showing as measured by the method of the present invention the gene encoding the interferon-gamma, a relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

FIG. 13 is as measured by the method of the present invention the gene coding for the interleukin-10 is a graph showing the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 14 is a graph showing as measured by the method of the present invention the gene encoding interleukin -12p35 subunit, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 15 is a graph showing as measured by the method of the present invention the gene encoding interleukin -12p40 subunit, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 16 is, as measured by the method of the present invention the gene coding for the interleukin-2 is a graph showing the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 17 is as measured by the method of the present invention the gene coding for the interleukin-10 is a graph showing the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 18 is a graph showing as measured by the method of the present invention the gene encoding interleukin -12p35 subunit, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 19 is a graph showing as measured by the method of the present invention the gene encoding interleukin -12p40 subunit, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/57 C07K 14/57 C12N 15/09 G01N 33/50 P G01N 33/50 C12N 15/00 A // (C12Q 1 / 68 C12R 1:91) (72) Inventor Hideyuki Ichikawa 1050 Nippa-cho, Kohoku-ku, Yokohama-shi, Kanagawa Pref.

Claims (13)

[Claims] When measuring the expression of a specific cytokine gene in a sample cell population, cells of the test cell population are crushed or lysed, mRNA is extracted from the treated cell, and cDNA is extracted from the mRNA. Synthesizing and said c
In a method of performing measurement by amplifying DNA, a cDD is obtained by adding mRNA as an external standard to the sample cell population or the cell processed product or mRNA extracted therefrom.
NA is synthesized and the cDNA preparation is split and one cD
Amplify the cytokine DNA from the NA preparation,
A method comprising amplifying an external standard DNA from a DNA preparation and comparing the two. 2. A method for measuring an increase in the expression level of a specific cytokine gene in a sample cell population by a test stimulator, wherein the cell population stimulated with the test stimulator and the test stimulator are stimulated. A cell population that has not been stimulated with the test stimulant, and a result obtained from the cell population stimulated with the test stimulator by performing the measurement according to claim 1 for each cell population. Comparing the results obtained from the method. 3. The method according to claim 1, wherein the amplification of the cDNA is performed by polymerase chain reaction (PCR). 4. The glyceraldehyde-3-phosphate-dehydrogenase (G
The method according to any one of claims 1 to 3, wherein mRNA of the (APDH) gene is used. 5. The method according to any one of claims 1 to 4, wherein rat or mouse mRNA is used as the GAPDH gene mRNA. 6. The mRNA of the rat GAPDH gene
In the PCR amplification, an oligonucleotide that hybridizes to the region from position 117 to position 136 in SEQ ID NO: 1 was used as one primer, and was hybridized to the region from position 266 to position 284 in SEQ ID NO: 1 as the other primer. The method according to claim 5, wherein an oligonucleotide that hybridizes to the region at positions 234 to 258 in SEQ ID NO: 1 is used as a probe, and an oligonucleotide that hybridizes to the region at positions 234 to 258 in SEQ ID NO: 1 is used as the probe. 7. The mRNA of the mouse GAPDH gene
In the PCR amplification, an oligonucleotide that hybridizes to the region from position 92 to position 111 in SEQ ID NO: 2 was used as one primer, and was hybridized to the region from position 241 to position 259 in SEQ ID NO: 2 as the other primer. An oligonucleotide that hybridizes to the region from nucleotide positions 209 to 234 in SEQ ID NO: 2 as a probe, or an oligonucleotide that hybridizes to the region from nucleotides 46 to 67 as one primer. And nucleotides 252-27 as the other primer
Oligonucleotides that hybridize to region 1 were used, and nucleotides 222 to 24 were used as probes.
The method according to claim 5, wherein oligonucleotides in two regions are used. 8. As a forward primer, 5′-
Using DNA using the nucleotide sequence of GTTACCAGGGCTGCCTTCTC-3 ′ (SEQ ID NO: 3), as a reverse primer,
5′-GGGTTTCCCGTTGATGACC-3 ′ (SEQ ID NO: 4) DNA using the base sequence and, as a probe, DNA having a sequence of 5′-AACGGCACAGTCAAGGCTGAGAATG-3 ′ (SEQ ID NO: 5); or The method according to claim 6, wherein primers and probes that have been modified by substitution, deletion and / or addition of 4 or less nucleotides in the sequence of the above, and that can hybridize to the corresponding region are used. 9. GTCACC as a forward primer
DNA using the sequence of AGGGCTGCCATTTG (SEQ ID NO: 6)
Using TGTCATCAACGGGAAG as a reverse primer
DNA having the base sequence of CCC (SEQ ID NO: 7) was used, and AACCGGCACAGTCAAGGCCGAGAATGG was used as a probe.
Using a DNA having the nucleotide sequence of (SEQ ID NO: 8); or 5'AATGGTGAAG as a forward primer
Using the base sequence of GTCGGTGTGAAC3 '(SEQ ID NO: 41), 5' GAAGATGGTGATGGGCTT was used as a reverse primer.
Using CC3 '(SEQ ID NO: 42) and 5'CAAGCTTCCCATTCTCGGCC3' (SEQ ID NO: 43) as a probe; or modified by substitution, deletion and / or addition of up to 4 nucleotides in said sequence. 8. The method of claim 7, wherein primers and probes are used that are capable of hybridizing to the corresponding region. 10. The method according to claim 1, wherein the specific cytokine gene is an interleukin gene or an interferon gene. 11. The specific cytokine gene is interleukin-2 gene, interferon-γ gene, interleukin-10 gene, interleukin-
The method according to claim 10, which is a 12p35 subunit gene or an interleukin 12p40 subunit gene. 12. The amplification of the cDNA using a forward primer and a reverse primer, and a probe that has a reporter and a quencher and hybridizes with a template nucleic acid in a region sandwiched between the two primers,
The method according to any one of claims 1 to 11, wherein the method is performed by performing PCR using a DNA polymerase having 5 'to 3' exonuclease activity. 13. The forward primer and the reverse primer according to any one of claims 6 to 9,
And a kit for measuring cytokine gene expression, comprising a probe.