RU2522801C2 - Method for detecting transcription level of gene coding human chemokine ccl2 (mcp-1) and kit for implementing it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. A method for detecting an expression level of MCP1 (CCL2) gene provides assessing its iRNA amount by a polymerase chain reaction with reverse transcription (RT-PCR) with real-time signal detection in human sampled cells, tissues and biological fluids. What is developed is a kit for quantitation of iRNA of MCP1 involving combinations of oligonucleotide primers and fluorescent probes for real-time RT-PCR to detect an amount of cDNA of MCP1 genes and RPL32, ACTB, PII normalisation genes.

EFFECT: using the group of inventions enables reducing calculation errors introduced when using one normalisation gene without increasing the number of amplifications, as well as reducing calculation errors raised in a simplified suggestion of the efficacy balance of the cDNA amplification reactions of MCP1 and a normalisation gene observed in ∆∆Ct method.

2 cl, 2 dwg, 3 tbl, 1 ex

Description

The present invention relates to medicine, in particular immunology, allergology and molecular biology, and relates to a method for determining the level of transcription of a gene encoding a human chemokine CCL2 (MCP-1) by RT-PCR with real-time detection of results, and a kit for its implementation .

The RT-PCR method with real-time detection of results is widely used in laboratory diagnostics and research. The main application of the method is the quantitative determination of mRNA molecules in a sample, including the determination of the level of mRNA expression of genes encoding human cytokines.

The RT-PCR method includes three stages: synthesis of cDNA molecules (DNA, complementary to cellular RNA) using the reverse transcription reaction; amplification of the cDNA sequence corresponding to the fragment of the analyzed gene; analysis of the results and determination of the relative amount and level of cDNA expression of the analyzed gene in the sample. The amount of specific cDNA in the sample, on the one hand, depends on the level of mRNA expression of the corresponding gene in the cell, on the other hand, it is determined by the volume of cellular material in the sample, the degree of degradation of the analyzed RNA, and the efficiency of the reverse transcription reaction.

The above factors are difficult to standardize for all analyzed samples. For example, the reverse transcription reaction is extremely sensitive to contaminant contaminants in the sample, which can lead to 5-90% fluctuations in the reaction efficiency [Ferre FA, Marchese P., Pezzoli S., Griffin E., Buxton H., Boyer V. Quantative PCR : an overview. // Polymerase Chain Reaction. / K.B. Mullis, Ferre F. R.A. Gibbs. - Burkhauser, Boston, 1994. - P.67-88]. In order to take into account the influence of additional factors on the amount of cDNA of the analyzed gene, the so-called “normalization coefficient” is calculated for each of the samples. When calculating the “normalization coefficient” in each sample, in addition to the amount of cDNA of the analyzed gene, the amount of cDNA of the “normalization gene” is determined. As “normalization genes”, genes are chosen for which the expression level in the cells of various samples is not presumably changed. Under this condition, a change in the amount of cDNA of the “normalization gene” will depend mainly on the influence of additional factors determining the kinetics of the reverse transcription reaction.

As a rule, genes of the “household” possess a similar property. As has been shown in publications, b- and g-actin genes can be used for normalization [Song Q., Wei T., Lees-Miller S., Ainemri E., Walters D., Lavin M.F. Resistance ofactin to cleavage during apoptosis. // Proc Natl Acad Sci USA. - 1997. - N1. - P.157-62], cyclophilin [Jaschke A., Mi, H. Tropschug, M. Human T cell cyclophilinl8 binds to thiol-specific antioxidant protein Aopi and stimulates its activity. // J Mol Biol. - 1998. - N4. - P.63-69], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [Tarze A., Deniaud, A. Le Bras, M. Maillier, E. Molle, D. Larochette, N. Zamzami, N. Jan, G. Kroemer, G. Brenner, C. GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization. // Oncogene. - 2007. - N26. - P.2606-2620], hypoxanthine phosphoribosyltransferase (HRPT) [Foss D.L., Baarsch M.J., Murtaugh M.P. Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues. // Anim Biotechnol. - 1998. - N1. - P.67-78.], L32 ribosomal protein and other ribosomal proteins, 18S, 28S rRNA [Finnegan M.S., Goepel J.R., Hancock B.W., Goyns M.H. Investigation of the expression of housekeeping genes in non-Hodgkin's lymphoma. // Leuk Lymphoma. - 1993. - N10. - P.387-393.], Type II RNA polymerase (PII), TATA box binding protein (TBP) [Thellin O., Zorzi W., Lakaye B., De Borman B., Coumans B., Hennen G ., Grisar T., Igout A., Heinen E. Housekeeping genes as internal standards: use and limits // Journal of Biotechnology. - 1999. - N75. - P.291-295].

The disadvantage of the method of data normalization using one normalization gene is the significant variability of the analysis results with the wrong choice of the normalizing gene. There is no ideal normalization gene for which the expression level will remain constant regardless of the tissue and state of cells in the analyzed sample. The very concept of a constant level of gene expression is rather arbitrary. The expression pattern of many “normalization genes” may vary [Goldsworthy S.M., Goldsworthy T.L., Sprankle C.S., Butterworth B.E. Variation in expression of genes used for normalization of Northern blots after induction of cell proliferation. // Cell Prolif. - 1993. - N6. - P.511-518]. For example, the expression of genes such as RPL32 and G3PDH, often used to calculate the “normalization coefficient”, changes under the influence of mitogenic agents [Thellin O., Zorzi W., Lakaye B., De Borman B., Coumans B., Hennen G., Grisar T ., Igout A., Heinen E. Housekeeping genes as internal standards: use and limits // Journal of Biotechnology. - 1999. - N75. - R.291-295]. The expression of many housekeeping genes is tissue-specific, in particular, it was shown that the expression of the b-2-microglobulin (B2M) gene differs 112 times in fetal brain tissues and white blood cells. The ASTV gene is expressed 22 times more intensively in cardiomyocytes compared with fibroblasts [Schmittgen T.D., Zakrajsek B.A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. // Genome Biology. - 2002. - N3. - P.3401-3411]. In this regard, the choice of the “normalizing gene” is one of the most critical steps in the analysis. The most optimal approach is that in which the expression of several “normalizing genes” is analyzed simultaneously. In this case, the “normalization coefficient” is calculated as the geometric mean or arithmetic average of the relative amount of cDNA of all “normalization genes” [Tricarico C., Pinzani P., Bianchi S., Paglierani M., Distante V., Pazzagli M., Bustin SA , Orlando C. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. // Anal Biochem. - 2002. - N309. - P.293-300]. The use of this approach also has its drawbacks, associated primarily with the need to formulate a large number of RT-PCR reactions, which is extremely inconvenient in the context of clinically significant application of the method. In addition, this approach is sometimes impossible due to the extremely small amount of cellular material in the sample.

Another weak point of the RT-PCR method is the stage of analysis of the obtained measurements. In most cases, the level of cDNA expression is estimated relative to the level of expression of the normalization gene using the DDS1 method. In this case, the relative amount of mRNA is determined in accordance with equation (1)

$$\frac{X_{S\mathrm{one}}}{{\mathrm{<figure-callout\; id="2"\; label="X\; S"\; filenames="00000002.png,00000003.png"\; state="\{\{state\}\}">X\; </figure-callout>}}_S}=2^{-\Delta \Delta Ct}(\mathrm{one})$$

Where

ΔΔCt = (Ct _S1 -Ct _Norm1 ) - (Ct _S2 -Ct _Norm )

Ct is the value of the amplification cycle at the point of intersection of the kinetic curve of accumulation of the amplification product with the line of the threshold level of fluorescence. The threshold level of fluorescence is determined automatically by the instrument software;

Ct _S1 and Ct _S2 - value of the threshold cDNA amplification cycle of the analyzed gene in two compared samples s1 and s2;

Ct _Norm1 and Ct _Norm2 are the values of the threshold cycle of amplification of the normalization gene cDNA in two compared samples s1 and s2.

Using the ΔΔCt method is possible provided that the efficiency of the cDNA amplification reactions of the analyzed and normalizing genes are equal and close to 100%. Failure to do so will result in significant errors in the results. However, in practice it is very difficult to achieve this condition.

The problem solved by the proposed invention is to eliminate the above disadvantages and create a more reliable and quick method for determining the level of transcription of the MCP1 gene, which is also applicable in clinical practice.

The proposed method for determining the level of transcription of the MCP1 gene by RT-PCR with real-time detection of the results involves: isolation of RNA from samples of the analyzed material (blood, tissues, scrapings, punctate, cell cultures), cDNA synthesis using the reverse transcription reaction, amplification of cDNA genes using real-time PCR. A kit for implementing the method is proposed, characterized by the use of specific oligonucleotide primers and fluorescence probes, as well as a set of normalization genes (RPL32, ASTV, PII), selected for the most correct calculation of the level of CCL2 (MCP1) in RNA samples. The results obtained are normalized using a normalization coefficient calculated as the arithmetic mean of the amount of cDNA of the RPL32, ASTV and PII genes encoding ribosomal protein L32, b-actin and polymerase-II, respectively. In this case, the cDNA amplification of the RPL32, ASTV and PII genes is carried out using one triplex-PCR.

When determining the relative amount of cDNA of the MCP1 gene and normalization genes RPL32, ASTV and PII, a calibration curve is used that was constructed by amplification of standard DNA samples taken in 6 successively decreasing triplicates of known concentrations. The standard is a mixture of DNA fragments carrying an amplifiable cDNA sequence of the RPL32, ASTV, PII, and MCP1 genes of 500 nucleotide pairs in length. The relative amount of cDNA of each gene is determined based on the equation of the calibration curve, taking into account the efficiency of amplification of each of the amplification reactions.

The technical result achieved using the proposed method is:

firstly, to reduce the calculation error introduced when using one normalization gene without increasing the number of amplifications;

secondly, in reducing the calculation error arising under the simplified assumption that the amplification reactions of the MCP1 cDNA amplification reactions are equal to the normalization gene that occurs in the ΔΔCt method.

Samples for analysis

Whole blood, mononuclear blood cells, biopsy samples, punctate, mucosal scrapings, cell cultures can be used as samples for analysis.

Isolation of RNA from samples.

Methods for isolating total RNA from mammalian tissue samples include the following stages: grinding samples in a homogenizer or in liquid nitrogen (refers to dense, difficult to homogenize tissue fragments), cell lysis, RNA isolation and purification, RNA quality control by electrophoresis in 1% agarose gel in the presence of dye ethidium bromide, as well as spectrophotometric determination of the amount of RNA. Homogenization of tissue pieces can be carried out manually, grinding with a pestle in a ceramic mortar, or using mechanical homogenizers, for example TissueLyser (Qiagen). Various protocols can be used for cell lysis and RNA isolation. In classical methods, strong chaotropic agents are used in combination with detergents, such as guanidine isothiocyanate and sarcosyl, for subsequent cell lysis and RNA isolation, followed by sequential extraction with phenol at pH5 5.0 and chloroform to denature and remove proteins and genomic DNA [Chomczynsci P., Sacci N. Single-step method of RNA isolation by Acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 1987 / Vol 162. P 156-159]. RNA is then precipitated in the presence of isopropanol or ethanol or sorbed on a solid support, for example Silica S-5631 (Sigma) or spin column (Qiagen; Promega). In order to prevent RNA degradation by RNases, ribonuclease inhibitors, such as, for example, recombinant RNAsin (Promega), can be used. To eliminate impurities of genomic DNA, it is possible to treat the resulting RNA preparation with DNase purified from ribonuclease, however, with a small amount of material, this step is best avoided to prevent possible loss and degradation of RNA. RNA isolation can be performed using the Trizol® Reagent [Invitrogen] method or using commercially available kits such as RNeasy kits (Qiagen), SV Total RNA Isolation System (Promega), etc.

Synthesis of cDNA using a reverse transcription reaction.

As a result of the reverse transcription reaction on the RNA matrix, a single-stranded DNA chain is synthesized. The resulting cDNA molecules are more stable compared to unstable RNA molecules and can be amplified by polymerase chain reaction to the amounts necessary for detection.

The reverse transcription reaction can be carried out using various commercial reverse transcriptase preparations, for example, Moloni mouse leukemia virus (M-MLV), avian myeloblastosis virus (AMV) reverse transcriptase, PowerScript (M-MLV-RT point mutation), C. Therm Polymerase, etc. Thermostable Thermus thermophilus (Tth) DNA polymerase, which has reverse transcriptase activity in the presence of Mn ²⁺ ions, can be used.

The reverse transcription reaction begins with the formation of a double-stranded seed, for this you can use various types of primers. For example, oligo (dT) n primers (n is usually 12-18) that bind to the endogenous polyA tail at the 3'-end of the mRNA. These primers should be used to obtain full-sized cDNA. Nucleotides A, C or G can be added to the 3'-end of the oligo (dT) sequence to “anchor” the primer at the border of the transcript and the poly-A tract.

Another variant of the primers is random hexanucleotide primers (statistical primers) that hybridize in several regions throughout the RNA. In the reverse transcription reaction using statistical primers, shorter (on average about 500 bp) cDNA fragments are synthesized. The reverse transcription reaction using statistical primers, as a rule, proceeds more efficiently, especially during reverse transcription of GC-rich regions and 5'-regions of mRNA.

In addition to hexamer primers, longer 9-12-dimensional primers can also be used. You can also use statistical primers in combination with oligo (dT) primers or specific primers complementary to the sequence of the analyzed gene.

Amplification of cDNA of RPL32, ASTV, PII, and MCP1 genes using real-time PCR.

Amplification of fragments of transcripts of the RPL32, ASTV, PII, and MCP1 genes is performed using pairs of primers complementary to transcripts. When choosing primers for amplification of PII and MCP1 cDNA, it is necessary to take into account the exon-intron structure of the gene and place the primers so that their hybridization sites are located in different exons. Thus, suppression of possible impurities of genomic DNA in the sample is achieved. This rule does not have to be followed when choosing primers for amplification of RPL32 cDNA and ASTV, since for these genes the human genome contains a large number of pseudogenes. In this case, when choosing primers, it is necessary to take into account the existing polymorphism of genes and pseudogenes RPL32 and ASTV in order to minimize hybridization of the primers with the sequence of pseudogenes.

The amplification reaction can be performed using a commercially available Thermus aquaticus DNA polymerase preparation, for example, HS Taq DNA polymerase, (Eurogen), GoTaq® Hot Start Polymerase (Promega), etc. MCP1 cDNA amplification is performed using a pair of primers complementary to the MCP1 transcript sequence The amplification of cDNA of the RPL32, ASTV and PII genes is performed in one tube using simultaneously three pairs of primers complementary to the sequences of RPL32, ASTV and PII transcripts. As a matrix, cDNA synthesized on total cellular RNA is used.

The accumulation of MCP1 amplification products is detected in real time using one of the methods:

- or by using a double-stranded DNA intercalating fluorescent dye with a high fluorescence discrimination index in the bound and unbound forms, for example SYBR GreenI (Invitrogen);

- or using an oligonucleotide sample of the TaqMan type complementary to the central part of the amplified transcript fragment. A sample of 20-30 nucleotides in length carries a fluorophore (FAM, R6G, ROX) at the 5'-end and a non-fluorescent quencher at the 3'-end (BHQ, FTQ, etc.). At each amplification cycle, the gabridized sample is hydrolyzed from the 5'-end, while the fluorophore is spatially disconnected from the quencher and, as a result, the fluorescence of the reaction mixture increases.

The accumulation of triplex amplification products of cDNA of the RPL32, ASTV and PII genes is detected using three oligonucleotide probes of the TaqMan type, complementary to the central part of the amplified fragments of RPL32, ASTV and PII transcripts.

Available software such as OligoAnalyser v. Can be used to select primers and samples of TaqMan type. 1.0.3, BeaconDisigner v. 7.0 and others. The use of the following primer pairs is preferred:

CCL2-F 5'-TCGCCTCCAGCATGAAAGTC-3 '

CCL2-R 5'-TCTGCACTGAGATCTTCCTATTG-3 '

CCL2-pr 5'-FAM-CCCTTCTGTGCCTGCTGCTCATAG-3 '

ACTB_F 5'-GTGCGTGACATTAAGGAGAAG-3 '

ACTB_R 5'-GAAGGAAGGCTGGAAGAGTG-3 '

ACTB_pr 5'-R6G-CGCCCTGGACTTCGAGCAAGAGA-BHQ-3 '

RPL32_F 5'-CAACATTGGTTATGGAAGCAACA-3 '

RPL32_R 5'-TGACGTTGTGGACCAGGAACT-3 '

RPL32_pr 5'-FAM-ACATGCTGCCCAGTGGCTTCCG-BHQ-3 '

POLR2A_F 5'-GCACCACGTCCAATGACAT-3 '

POLR2A_R 5'-GTGCGGCTGCTTCCATAA-3 '

POLR2A_pr 5'-ROX-TACCACGTCATCTCCTTTGATGGCTCCTAT- BHQ-3 '

The determination of the amount of cDNA of the RPL32, ASTV, PII, and MCP1 genes is performed using the equation of the calibration curve taking into account the effectiveness of each amplification reaction. To construct a calibration curve, standard DNA samples with a known successively three-fold decrease in DNA concentration are amplified simultaneously with the analyzed samples. The standard sample is a 500 bp double-stranded DNA mixture containing sequences of amplified fragments of RPL32, ASTV, PII, and MCP1 transcripts. The calibration curve is plotted against the Ct and Log values of the amount of DNA in the standard sample. In accordance with the obtained graph of the calibration curve using regression analysis, the coefficients of the linear equation of the calibration curve are determined. The resulting equation is used to determine the amount of cDNA RPL32, ASTV, PII and MCP1 in the analyzed samples.

Determination of the transcription level of cDNA of the MCP1 gene is carried out by normalizing the amount of MCP1 cDNA in the sample. For this, a normalization coefficient is calculated. The normalization coefficient is calculated as the arithmetic mean of the amount of cDNA RPL32, ASTV, PII in the sample.

To perform real-time PCR, various companies have developed amplifiers, for example, ABI Prism 7000 Sequence Detection System (Applied Biosystems), CFX96 (Bio-Rad), and domestic devices DT-322 (DNA-Technology). The use of PCR with the detection of results in real time can reduce the risk of contamination and automate the diagnostic process. The reaction takes from 40 minutes to two hours (depending on the amplifier used) and includes simultaneous PCR, detection of a fluorescent signal, data processing and their presentation in graphical form using special software.

The invention is as follows.

Example 1

The research material was peripheral blood samples of healthy donors and patients with atopic dermatitis in the acute stage. Samples of peripheral blood with a volume of 5 ml were collected in tubes containing 0.1 M EDTA solution (anticoagulant that prevents blood coagulation).

Isolation of Mononuclear Blood Cells (MCC). The separation of the cellular elements of the blood was carried out by centrifugation in a density gradient of ficoll-verografin (density 1.077 g / ml). The technique of isolation of blood lymphocytes on ficoll [Boyum A. Separation of leukocytes from blood and bone marrow // Scand. J. Clin. Lab. Investig. - 1968. - Vol.21. - Suppl. 97 - p. 1-9]. The resulting cells were resuspended in RPMI culture medium containing 10% FBS and 0.03% glutamine at a concentration of 10 ⁶ cells / ml and plated in 1000 μl / well in the 24-well culture plate. Cells were cultured for 24 hours at 37 ° C and 5% CO ₂ in the atmosphere.

RNA isolation and cDNA synthesis in a reverse transcription reaction. Cells after 24 h of cultivation were freed from conditioned media by centrifugation at 600 g and lysed with Trisol reagent (Invitrogen). Total cellular RNA was isolated according to the recommendations of Invitrogen. The quality of RNA preparations was evaluated by electrophoretic separation in a 1.3% agarose gel. The reverse transcription reaction was carried out at 42 ° C for 45 min in 20 μl of the reaction mixture containing 10 mm Tris-HCl (pH 8.3), 5 mm MgCl ₂ , 10 mm DTT, 50 mm KCl, 0.2 mm dNTP, Stat-9 primer (10 ng / μl), 100 units Act. MoMLV DNA-dependent RNA polymerase (Biosan, SORAN ICBPM) and 500 ng of total cellular RNA.

Amplification of cDNA of the MCP1 gene with real-time detection. The amplification reaction was carried out using thermal cyclers with an optical unit for iQ5 iCycler or CFX96 fluorescence detection (Bio-Rad). The possibility of real-time fluorescence detection was achieved by adding SYBRGreen I intercalating dye to the reaction mixture (method 1) or using TaqMan type hybridization samples (method 2).

Method 1. PCR was carried out in a volume of 25 μl, the reaction mixture contained 10 mm Tris-HCl (pH 8.9); 55 mM KCl; 0.05% Tween 20; 2.5 mm MgCl ₂ , 0.2 mm dNTP, 300 mm primers (forward and reverse), 0.5 units Act. Taq DNA polymerase (Biosan, ICBPM SORAN), SYBRGreen I [1: 25000] and 0.1-5 ng cDNA.

For amplification, primers were used: forward primer - 5'-TCGCCTCCAGCATGAAAGTC-3 'and reverse primer - 5'-TCTGCACTGAGATCTTCCTATTG-3'.

Amplification Protocol:

- primary denaturation - 3 minutes at 96 ° C

- amplification cycle (× 40)

- denaturation - 10 seconds at 96 ° C

- primer annealing - 10 seconds at 60 ° C

- elongation - 10 seconds at 72 ° C

- signal pick-up - 10 seconds at 86 ° C (melting point of the amplification product)

- building a melting curve - heating the amplification mixture from 75 to 95 ° C, in increments of 0.5 ° C, accompanied by a fluorescence signal at each step for 10 seconds.

The specificity of amplification was confirmed by analysis of the melting curves, as well as by electrophoretic analysis in an 8% polyacrylamide gel.

Method 2. PCR was carried out in a volume of 25 μl, the reaction mixture contained 65 mm Tris-HCl (pH 8.9); 24 mM (NH ₄ ) ₂ SO ₄ ; 0.05% Tween 20; 2.5 mM MgCl ₂ , 0.2 mM dNTP, 300 mM primers (forward and reverse), 100 mM Taq-Man assay, 0.5 unit Act. Taq DNA polymerase (Biosan, ICBPM SORAN) and 0.1-5 ng cDNA.

Amplification Protocol:

- primary denaturation - 3 minutes at 96 ° C

- amplification cycle (× 40)

- denaturation - 10 seconds at 96 ° C

- annealing of primers, elongation and removal of the fluorescent signal - 60 seconds at 60 ° C.

The specificity of amplification was confirmed by polyacrylamide gel electrophoretic analysis.

For amplification, oligonucleotide samples were used: direct primer - 5'-TCGCCTCCAGCATGAAAGTC-3 'and reverse primer - 5'-TCTGCACTGAGATCTTCCTATTG-3', hybridization probe - 5'-FAM-CCCTTCTGTGCCTGCTGCTC.

Amplification of cDNA of RPL32, ASTV and PII genes with real-time detection. PCR was carried out in a volume of 25 μl, the reaction mixture contained 65 mm Tris-HCl (pH 8.9); 24 mM (NH ₄ ) ₂ SO ₄ ; 0.05% Tween 20; 2.5 mM MgCl ₂ 0.2 mM dNTP, 300 mM primers (RPL32_F and RPL32_R, ACTB_F and ACTB_R, PII_F and PII_R), 100 mM Taq-Man samples (RPL32_pr, ACTB_pr, PII_pr), 0.5 units. Act. Taq DNA polymerase (Biosan, ICBPM SORAN) and 0.1-5 ng cDNA.

Amplification Protocol:

- primary denaturation - 3 minutes at 96 ° C

- amplification cycle (× 40)

- denaturation - 10 seconds at 96 ° C

- annealing of primers, elongation and removal of the fluorescent signal - 60 seconds at 60 ° C.

The specificity of amplification was confirmed by polyacrylamide gel electrophoretic analysis.

For amplification, oligonucleotide samples were used:

ASTV: direct primer 5'-GTGCGTGACATTAAGGAGAAG-3 ', reverse primer 5'-GAAGGAAGGCTGGAAGAGTG-3', hybridization test 5'-R6G-CGCCCTGGACTTCGAGCAAGAGA-BHQ-3 '

RPL32: forward primer 5'-CAACATTGGTTATGGAAGCAACA-3 ', reverse primer 5'-TGACGTTGTGGACCAGGAACT-3', hybridization probe 5'-FAM-ACATGCTGCCCAGTGGCTTCCG-BHQ-3 '

PII: direct primer 5'-GCACCACGTCCAATGACAT-3 ', reverse primer 5'-GTGCGGCTGCTTCCATAA-3', hybridization test 5'-ROX-TACCACGTCATCTCCTTTTGATGGCTCCTAT-BHQ-3 '.

Analysis of the results of RT-PCR.

A total of 48 samples were analyzed, of which 24 samples of MCC obtained from healthy donors, and 24 samples of MCC from patients with atopic dermatitis. The relative amount of cDNA RPL32, ASTV, PII and MCP1 in the analyzed samples was determined using the equation of the calibration curve. For this, 5 standard samples were amplified simultaneously with the analyzed samples with a DNA concentration successively decreasing by three times (st1-1 y.e., st2-3 y.e., st3-9 y.e., st4-27 y.e., st5-81 y.e.). For each sample, the threshold cycle value (Ct) was determined using the instrument software at which the fluorescence value of the reaction mixture reached a certain “threshold” level (T) that was the same for all compared samples.

The amount of cDNA (X ₀ ) in the sample was calculated using the equation:

Log (X ₀ ) = a + b * Ct,

where a = log (T) and b = log (1 + Eff).

The amplification conditions were chosen so that the efficiency (Eff) of PCR remained constant for all analyzed samples and samples of the calibration curve, therefore, the coefficient b = log (1 + E) = const.

The coefficients a and b of the equation were calculated when constructing the equation of the calibration curve. The calibration curve equation determined the relationship between the Ct values obtained from the amplification of five standard samples and the log (X ₀ ) values of these samples (in our case, these are Igl, log3, log9, log27, log81).

On figa shows curves of increasing the fluorescence intensity of the reaction mixture during amplification of a fragment of the transcript MCP1 for 5 standard samples with successively decreasing three times the concentration of DNA depending on the number of PCR cycles. Amplification was performed by method 2 using a pair of primers and TaqMan hybridization assay. On figb shows a graph of the calibration curve that determines the dependence of the Ct values obtained by amplification of five standard samples, and the values of lg (X ₀ ) of these samples (lg1, lg3, lg9, lg27, lg81).

Amplification efficiency was determined as the angle of inclination of the calibration curve. For all pairs of primers, the amplification efficiency was not less than 90%. The values of the efficiency of the amplification reactions of cDNA RPL32, ASTV, PII and MCP1 are shown in table 1.

To assess the reproducibility of the threshold cycle (Ct) values, all analyzed samples with each pair of primers were amplified in triplicate. The difference in the Ct values between repetitions was no more than 0.3 cycles.

To determine the relative level of MCP1 cDNA expression, the values of the relative amount of MCP1 cDNA obtained for the analyzed samples were normalized using a normalization coefficient calculated as the arithmetic mean of the amount of cDNA of the RPL32, ASTV, PII genes

Normalization genes should be characterized by a constant level of expression in the cells and not depend on the analyzed sample. In order to assess the variability of the level of expression of the RPL32, ASTV, PII genes between samples, pairwise correlation coefficients (R ² ) between the amount of RPL32 cDNA and ASTV, RPL32 and PII, ASTV and PII were calculated. The values of the pairwise Pearson correlation coefficients (R ² ) of the cDNA quantities of the normalizing genes RPL32, ASTV, PII are presented in Table 2. In all cases, high correlation coefficients of at least 0.73 were obtained.

Comparison of the results obtained when determining the level of transcription of MCP1 cDNA by RT-PCR and the amount of MCP1 protein by ELISA.

Assessment of the level of MCP1 protein production by mononuclear cells was carried out in conditioned culture media collected after centrifugation of cultured 24 hours cells. Aliquots of the samples were stored at -20 ° C until analysis.

The analysis of the MCP-1 level was carried out by the enzyme immunoassay (ELISA) using commercial sets of Vector-Best CJSC Novosibirsk, in strict accordance with the manufacturer's instructions. ELISA results were detected on a TriStar LB941 analyzer (Berthold Technology Inc.).

Figure 2 shows a graph of the dependence of the values of the expression level of MCP1 cDNA, determined by RT-PCR, and the values of the amount of MCP1 protein, determined by ELISA in 48 samples of mononuclear cell cultures. The values of the expression level of MCP1 cDNA obtained using the RT-PCR method were significantly correlated with the corresponding values of the amount of MCP1 protein determined by ELISA. The Pearson correlation coefficient R ² was 0.63, which corresponds to a level of statistical significance <0.001 (for the number of degrees of freedom n = 46).

Table 3 shows the results of a statistical analysis of the levels of MCP1 cDNA expression and the amounts of MCP1 protein in MCC samples from healthy donors and patients with atopic dermatitis in the acute stage. The analysis showed that the average value of the MCP1 cDNA expression level in MCC samples obtained from healthy donors is significantly higher than the MCP1 cDNA expression level in MCC samples of patients with atopic dermatitis in the acute stage (with significance level p <0.02).

ELISA results also indicate that MCC cells obtained from healthy donors produce a significantly larger amount of MCP1 protein than MCC cells in patients with atopic dermatitis (with significance level p <0.001) (table 3).

Table 1. Gene name PCR method Efficiency MCP1 amplification with SYBRGreenI 96.6 amplification with TaqMan breakdown 94.0 Rpl32 triplex-PCR with TaqMan samples 98.2 ASTV 95.5 Pii 92.7

Table 2. Rpl32 ASTV RII Rpl32 0.86 0.73 ACTS 0.91 Pii

Table 3. MCC healthy donors (n = 24) MCC of patients with atopic dermatitis (n = 24) Average value (conventional units) Standard deviation Average value (c.u.) Standard deviation The significance level of the unpaired t-test (P) the level of expression of the MCP1 cDNA 1.27 1,03 0.55 0.67 0.02 the amount of protein MCP1 0.93 0.47 0.34 0.41 <0.001

The list of oligonucleotide sequences for the application "Method for determining the level of transcription of a gene encoding a chemokine CCL2 (MCP-1) person, and a kit for its implementation."

A kit for determining the level of transcription of a gene encoding a human CCL2 chemokine (MCP-1) according to claim 1, including:

a) oligonucleotide primers and samples for amplification using one triplex-PCR cDNA of the RPL32, ACTB and PII genes encoding ribosomal protein L32, b-actin and polymerase-II, respectively

ACTB_F 5'-GTGCGTGACATTAAGGAGAAG-3 '

ACTB_R 5'-GAAGGAAGGCTGGAAGAGTG-3 '

ACTB_pr 5'-R6G-CGCCCTGGACTTCGAGCAAGAGA - BHQ- 3 '

RPL32_F 5'-CAACATTGGTTATGGAAGCAACA-3 '

RPL32_R 5'-TGACGTTGTGGACCAGGAACT-3 '

RPL32_pr 5'-FAM-ACATGCTGCCCAGTGGCTTCCG-BHQ- 3 '

POLR2A_F 5'-GCACCACGTCCAATGACAT-3 '

POLR2A_R 5'-GTGCGGCTGCTTCCATAA-3 '

POLR2A_pr 5'- ROX-TACCACGTCATCTCCTTTGATGGCTCCTAT- BHQ-3 '

b) oligonucleotide primers and a sample for amplification using PCR cDNA of the gene encoding CCL2 (MCP-1)

CCL2-F 5'-TCGCCTCCAGCATGAAAGTC-3 '

CCL2-R 5'-TCTGCACTGAGATCTTCCTATTG-3 '

CCL2-pr 5'-FAM-CCCTTCTGTGCCTGCTGCTCATAG-3 '.

Claims (2)

1. A method for determining the level of transcription of a gene encoding a human CCL2 chemokine (MCP1), including the isolation of RNA from samples of the analyzed material (blood, tissues, scrapings, punctate, cell cultures), cDNA synthesis using the reverse transcription reaction, amplification of cDNA genes using PCR in real time, characterized in that
a) the normalization of the results is carried out using a normalization coefficient calculated as the arithmetic mean of the cDNA sample of the RPL32, ASTV and PII genes encoding the ribosomal protein L32, b-actin and polymerase-II, respectively, while the cDNA amplification of the RPL32, ASTV and PII genes carried out using one triplex-PCR;
b) the accumulation of amplification products is detected in real time using one of the methods:
1b) using a double-stranded DNA intercalating fluorescent dye with a high fluorescence discrimination index in the bound and unbound forms, for example, SYBR GreenI (Invitrogen);
2b) using an oligonucleotide sample of the TaqMan type complementary to the central part of the amplified fragment of the transcript; a sample of 20-30 nucleotides in length carries a fluorophore (FAM, R6G, ROX) at the 5'-end and a non-fluorescent quencher at the 3'-end (BHQ, FTQ, etc.);
c) the determination of the relative amount of cDNA of the MCP1 gene and normalization genes RPL32, ASTV and PII is carried out using a calibration curve constructed by amplification of standard DNA samples taken in 6 successively decreasing triplicates of known concentrations; the standard is a mixture of DNA fragments carrying an amplifiable cDNA sequence of the RPL32, ASTV, PII, and MCP1 genes with a length of 500 nucleotide pairs; the relative amount of cDNA of each gene is determined based on the equation of the calibration curve, taking into account the amplification efficiency of each of the amplification reactions. 2. A kit for determining the level of transcription of a gene encoding a chemokine CCL2 (MCP1) of a person according to claim 1, including:
a) oligonucleotide primers and samples for amplification using one triplex-PCR cDNA of the RPL32, ASTV and PII genes encoding ribosomal protein L32, b-actin and polymerase-II, respectively
ACTB_F 5'-GTGCGTGACATTAAGGAGAAG-3 '
ACTB_R 5'-GAAGGAAGGCTGGAAGAGTG-3 '
ACTB_pr 5'-R6G-CGCCCTGGACTTCGAGCAAGAGA-BHQ-3 '
RPL32_F 5'-CAACATTGGTTATGGAAGCAACA-3 '
RPL32_R 5'-TGACGTTGTGGACCAGGAACT-3 '
RPL32_pr 5'-FAM-ACATGCTGCCCAGTGGCTTCCG-BHQ-3 '
POLR2A_F 5'-GCACCACGTCCAATGACAT-3 '
POLR2A_R 5'-GTGCGGCTGCTTCCATAA-3 '
POLR2A_pr 5'- ROX-TACCACGTCATCTCCTTTGATGGCTCCTAT-BHQ-3 '
b) oligonucleotide primers and samples for amplification using PCR cDNA of the gene encoding CCL2 (MCP1)
CCL2-F 5'-TCGCCTCCAGCATGAAAGTC-3 '
CCL2-R 5'-TCTGCACTGAGATCTTCCTATTG-3 '
CCL2-pr 5'-FAM-CCCTTCTGTGCCTGCTGCTCATAG-3 '.

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