An efficient and accurate qPCR detection method for blood gene expression levels

Abstract

The present invention discloses an efficient and accurate qPCR detection method for blood gene expression level, which relates to the field of biological detection technology. The method comprises the following specific steps: blood sample processing and RNA extraction: blood samples are collected under sterile conditions, and RNA is extracted by using an adapter kit through lysis, stratification, precipitation and washing operations. The present invention significantly reduces experimental errors and improves detection accuracy by optimizing experimental steps such as sample processing, reverse transcription reaction and fluorescent quantitative PCR reaction. In the sample processing stage, a total RNA extraction kit suitable for blood samples is used, and aseptic operation specifications are followed to effectively avoid sample contamination and RNA degradation. In the reverse transcription reaction and fluorescent quantitative PCR reaction stages, the detection method of the present invention can more accurately reflect the expression level of the target gene in the blood sample by accurately preparing the reaction solution, optimizing the reaction conditions and adopting a scientific data processing strategy.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an efficient and accurate qPCR detection method for blood gene expression levels. Background Art

[0002] In the field of biomedical research and clinical diagnosis, accurate measurement of gene expression levels in the blood is of great significance. This technology not only helps to deeply understand the occurrence and development mechanism of the disease, but also provides a key basis for early diagnosis of the disease, disease monitoring and the formulation of personalized treatment plans. With the continuous development of biotechnology, a variety of detection methods have emerged, aiming to achieve efficient and accurate detection of blood gene expression levels.

[0003] Although qPCR technology has been widely used in gene expression detection, the qPCR detection process for blood samples still faces some challenges. On the one hand, the traditional blood gene expression detection technology has a complex operation process, requires high professional skills of the experimenter, and the detection process is time-consuming, which makes it difficult to meet the needs of fast and efficient detection. On the other hand, some existing detection methods are insufficient in sensitivity and accuracy, and are easily affected by various factors such as impurities in the sample and changes in the experimental environment, resulting in poor reliability of the test results. In particular, in the sample processing, reverse transcription reaction system construction, and data processing and analysis, further optimization is still needed to improve the overall performance of the detection.

[0004] In view of the above problems, it is necessary to optimize the existing qPCR detection method of blood gene expression level, and realize efficient and accurate detection of blood gene expression level by optimizing experimental steps, precisely controlling reaction conditions and adopting data processing strategies. Therefore, it is of great significance to develop an efficient and accurate qPCR detection method of blood gene expression level that can comprehensively realize the above characteristics. Summary of the invention

[0005] The purpose of the present invention is to make up for the shortcomings of the prior art and provide an efficient and accurate qPCR detection method for blood gene expression levels. It can achieve efficient and accurate detection of blood gene expression levels by optimizing experimental steps, accurately controlling reaction conditions and adopting scientific data processing strategies. In the sample processing link, a total RNA extraction kit suitable for blood samples is used to effectively improve the extraction efficiency and purity of RNA. In the reverse transcription reaction and fluorescent quantitative PCR reaction links, the sensitivity and accuracy of the detection are significantly improved by accurately preparing the reaction solution, optimizing the reaction conditions and other measures. In addition, the present invention also adopts a scientific data processing strategy, using 2 -△△Ct The formula calculates the expression level of the target gene, further improving the reliability of the test results.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: an efficient and accurate qPCR detection method for blood gene expression level, the method comprising the following specific steps:

[0007] Blood sample processing and RNA extraction: Blood samples were collected under sterile conditions, and RNA was extracted using an adapter kit after lysis, stratification, precipitation, and washing. After being dissolved in an RNase-free solvent, its quality was tested by an ultraviolet spectrophotometer, and qualified samples were stored in an ultra-low temperature environment;

[0008] Construction of reverse transcription reaction system: Select a reverse transcription kit according to the target gene, thaw the reagent on ice, and prepare the genomic DNA removal reaction solution and reverse transcription PCR reaction solution according to the instructions of the reverse transcription kit;

[0009] Reverse transcription reaction: remove the genomic DNA reaction solution and incubate it, place it in a PCR instrument, and reverse transcribe the RNA into cDNA according to the temperature and time program recommended by the kit;

[0010] Preparation and reaction of fluorescent quantitative PCR reaction system: prepare reagents according to the optimized formula, including cDNA template, upstream and downstream primers, premixed reagents containing Taq polymerase, deoxyribonucleoside triphosphates, fluorescent dyes and reaction buffer, and sterile double distilled water, prepare the reaction solution on ice, mix well and put it into the reaction tube, seal and centrifuge it, put it into the fluorescent quantitative PCR instrument, complete the pre-denaturation, multiple rounds of cycle reaction and melting curve analysis according to the preset program, and control the temperature and time of each step;

[0011] Data processing and gene expression level calculation: After the reaction, the original data were obtained using the instrument software, exported and sorted, and outliers were removed. -△△Ct The formula was used to calculate △Ct and △△Ct based on the Ct values ​​of the target gene and the reference gene, thereby obtaining the ratio of the target gene expression levels between samples and evaluating the gene expression difference.

[0012] Furthermore, in the blood sample processing and RNA extraction step, 0.5-5 mL of blood sample is collected under sterile conditions, the collected blood sample is transferred to a lysis container provided by the kit, a matching lysis reagent is added, and the mixture is oscillated at an oscillation speed of 1000-3000 rpm for 5-15 minutes to fully lyse the blood cells, and then a stratification reagent is added, and the mixture is centrifuged at a speed of 10000-15000 rpm for 5-10 minutes for stratification. After the aqueous phase containing RNA is transferred to a new container, a precipitation reagent is added, the mixture is placed on ice for 10-30 minutes, and the mixture is centrifuged at a speed of 8000-12000 rpm for 10-20 minutes to precipitate the RNA, and the precipitate is washed 2-3 times with 70%-80% ethanol, and after each wash, the mixture is centrifuged at a speed of 7000-10000 rpm for 5-8 minutes, and 20-100 μL of RNase-free DEPC water is used to dissolve the precipitated RNA.

[0013] Furthermore, in the blood sample processing and RNA extraction steps, the absorbance of the RNA solution is measured at wavelengths of 260nm and 280nm using an ultraviolet spectrophotometer. When the A260 / A280 ratio is between 1.8-2.0, it indicates that the RNA purity is qualified. The RNA samples that have passed the quality inspection are stored in an ultra-low temperature refrigerator set at a temperature of -70°C to -80°C, and the samples are properly labeled and recorded.

[0014] Furthermore, in the step of constructing the reverse transcription reaction system, a genomic DNA removal reaction solution is prepared. Specifically, the operation is performed on ice, and 1-10 μL of RNA sample, 0.5-2 μL of genomic DNA contamination removal reagent, and 1-4 μL of 5×gDNA Eraser buffer are added to a centrifuge tube in sequence, and the total volume is supplemented with DEPC water to 10-20 μL. The components are evenly mixed by gently operating a pipette.

[0015] Furthermore, in the reverse transcription reaction system construction step, a reverse transcription PCR reaction solution is prepared. Specifically, 5-15 μL of genomic DNA removal reaction solution, 2-6 μL of 5×Primescript Buffer, 0.5-2 μL of reverse transcriptase 5×Primescript RT Enzyme Mix, and 2-6 μL of reverse transcription random primer RT Primer Mix are added in sequence on ice, and DEPC water is added to make the total volume 20-30 μL, and the components are evenly mixed by gentle operation with a pipette.

[0016] Furthermore, in the reverse transcription reaction step, the genomic DNA removal reaction solution is removed and incubated. Specifically, the prepared genomic DNA removal reaction solution is briefly centrifuged to concentrate the liquid in the tube at the bottom of the tube, the centrifuge tube is placed in an environment of 25°C-42°C for incubation for 5-15 minutes, and it is placed in a PCR instrument and reverse transcription is performed at 37°C-42°C for 10-60 minutes to synthesize cDNA from RNA under the action of reverse transcriptase, and an enzyme inactivation step is performed at 70°C-85°C for 3-10 minutes to terminate the reverse transcription reaction and obtain a high-quality cDNA product.

[0017] Furthermore, in the preparation and reaction steps of the fluorescent quantitative PCR reaction system, reagents are prepared according to an optimized formula, including 1-5 μL of cDNA template, 0.2-0.8 μL of PCR upstream primer with a concentration of 10 μM, 0.2-0.8 μL of PCR downstream primer with a concentration of 10 μM, 5-15 μL of a premixed reagent containing Taq polymerase, deoxyribonucleoside triphosphate, SYBR Green and reaction buffer, and sterile double distilled water is added to supplement the total volume to 15-30 μL.

[0018] Furthermore, in the preparation and reaction steps of the fluorescent quantitative PCR reaction system, the reagents are placed in a fluorescent quantitative PCR instrument, and pre-denaturation, multiple rounds of cycle reaction and melting curve analysis are completed according to a preset program, and the temperature and time of each step are controlled. Specifically, in the pre-denaturation stage, the temperature is maintained at 94°C-96°C for 5-15 minutes. In the cycle reaction stage, the denaturation temperature is set to 94°C-96°C for 1-5 seconds, the annealing temperature is 55°C-65°C for 10-30 seconds, the extension temperature is 70°C-75°C for 5-20 seconds, and the cycle is repeated 35-45 times. During the melting curve analysis, the temperature is maintained at 95°C for 10-30 seconds, the temperature is slowly reduced to 60°C-70°C, and then the temperature is increased to 95°C at a rate of 0.1°C-0.3°C / second.

[0019] Furthermore, in the data processing and gene expression level calculation steps, 2 -△△Ct Formula, △Ct and △△Ct are calculated based on the Ct values ​​of the target gene and the reference gene, so as to obtain the ratio of the target gene expression levels between samples. Specifically, CtA1 is set as the Ct value of the target gene of sample No. 1, CtB1 is set as the Ct value of the reference gene of sample No. 1, CtA2 is set as the Ct value of the target gene of sample No. 2, and CtB2 is set as the Ct value of the reference gene of sample No. 2. Then the ratio of the target gene expression levels of sample No. 1 and sample No. 2 is calculated as follows: △△Ct=(CtA2-CtB2)-(CtA1-CtB1)=X, then the expression level of the target gene in sample No. 2 is the 2nd level in sample No. 1. -X times.

[0020] Compared with the existing technology, this efficient and accurate blood gene expression level qPCR detection method has the following beneficial effects:

[0021] 1. The present invention significantly reduces experimental errors and improves detection accuracy by optimizing experimental steps such as sample processing, reverse transcription reaction and fluorescent quantitative PCR reaction. In the sample processing stage, a total RNA extraction kit suitable for blood samples is used, and aseptic operation specifications are followed to effectively avoid sample contamination and RNA degradation. In the reverse transcription reaction and fluorescent quantitative PCR reaction stages, the detection method of the present invention is able to more accurately reflect the expression level of the target gene in the blood sample by accurately preparing the reaction solution, optimizing the reaction conditions and adopting a scientific data processing strategy.

[0022] Second, the present invention optimizes the experimental steps and reaction conditions to make the entire detection process more concise and efficient, reducing unnecessary operation steps and time waste, and in terms of data processing, uses scientific data analysis software and -△△Ct The formula can be used for calculation to quickly and accurately obtain the expression level of the target gene, greatly shortening the time for data processing and analysis, so that the detection method of the present invention can meet the needs of rapid detection and provide a more convenient and efficient detection means for clinical diagnosis and biomedical research.

[0023] Other advantages, objectives and features of the present invention will be set forth in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 It is a schematic diagram of the structure of an efficient and accurate qPCR detection method for blood gene expression levels;

[0026] Figure 2 This is a flow chart of an efficient and accurate qPCR detection method for blood gene expression levels;

[0027] Figure 3 Schematic diagram of the changes in the expression of circ104600, ID2 and miR-592. DETAILED DESCRIPTION

[0028] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0029] Embodiment 1

[0030] according to Figure 1 The structure shown, and Figure 2 The process shown in the figure selects 50 liver cancer patients and 50 healthy controls. Under strict aseptic operation, 3 mL of venous blood samples are collected from each subject, collected using anticoagulant tubes, and TAKARA PrimeScript TM RT reagent Kit with gDNA Eraser reagents, transfer the collected blood sample to the lysis container provided by the kit, add the lysis reagent, oscillate at 2000rpm for 10 minutes to fully lyse the blood cells, add the stratification reagent, centrifuge at 12000rpm for 8 minutes to stratify, carefully transfer the aqueous phase containing RNA to a new container, add the precipitation reagent, place on ice for 20 minutes, then centrifuge at 10000rpm for 15 minutes to precipitate the RNA, wash the precipitate with 75% ethanol 3 times, centrifuge at 8000rpm for 6 minutes after each wash, finally, use 50μL of ribonuclease-free DEPC water to dissolve the precipitated RNA, use a UV spectrophotometer to measure the absorbance of the RNA solution at 260nm and 280nm wavelengths, ensure that the A260 / A280 ratio is between 1.8-2.0, and store the qualified RNA samples in an ultra-low temperature refrigerator at -80℃.

[0031] A kit suitable for reverse transcription of the circ 104600 gene was selected and operated on ice. First, a reaction solution for removing genomic DNA was prepared. 5 μL of RNA sample, 1 μL of genomic DNA contamination removal reagent, and 2 μL of 5×gDNAEraser buffer were added to a centrifuge tube, and DEPC water was added to make the total volume 15 μL. After thorough mixing, a reverse transcription PCR reaction solution was prepared. 10 μL of the first step reaction solution was taken, 4 μL of 5×Primescript Buffer, 1 μL of 5×Primescript RT EnzymeMix, and 3 μL of RT Primer Mix were added, and the total volume was made up to 25 μL with DEPC water. After gentle mixing, the reaction solution for removing genomic DNA was incubated at 37°C for 10 minutes, and then the reaction tube was briefly centrifuged and placed in a PCR instrument. Reverse transcription PCR reaction was performed according to the program of 37°C for 30 minutes and 85°C for 5 minutes to obtain the cDNA of the circ104600 gene.

[0032] Prepare the fluorescence quantitative PCR reaction system, add 2 μL cDNA template, 0.4 μL PCR upstream primer (10 μM), 0.4 μL PCR downstream primer (10 μM), 10 μL premixed reagent containing Taq polymerase, deoxyribonucleoside triphosphate, SYBR Green dye and reaction buffer to the PCR reaction tube on ice, then add 7.2 μL sterile double distilled water to a total volume of 20 μL, mix the reaction solution to avoid bubbles, if there are bubbles, centrifuge briefly to remove them, seal the reaction tube and centrifuge it instantly to make the reaction solution evenly distributed at the bottom of the tube, put it into the fluorescence quantitative PCR instrument, and react according to the procedure of pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 2 seconds, annealing at 60°C for 20 seconds, and extension at 70°C for 10 seconds, and finally perform melting curve analysis (95°C for 15 seconds, 70°C for 1 minute, 95°C for 15 seconds).

[0033] After the reaction, data processing and analysis were performed. The original data were obtained using the instrument's own software and exported to a spreadsheet for organization. The expression level of circ 104600 gene was calculated by the formula. The samples of liver cancer patients were set as the experimental group and the samples of healthy controls were set as the control group. The Ct values ​​of the target gene and the reference gene of the two groups of samples were determined respectively. △Ct and △△Ct were calculated, and then the ratio of the expression level of circ 104600 gene between the samples was obtained. The two groups of data were analyzed by T.test statistical method. The research found that Figure 3 As shown in the results, compared with healthy controls, the expression level of circ104600 gene in the blood of liver cancer patients was significantly upregulated (P<0.05). This result suggests that circ 104600 gene plays an important role in the occurrence and development of liver cancer. Figure 3 In the middle, * indicates various statistical calculations relative to the control group, * indicates P < 0.05, and ** indicates P < 0.01.

[0034] Embodiment 2

[0035] Experimental animals (such as mice) at a specific stage of embryonic development were selected, with 10 mice in each group. Under sterile conditions, 2 mL of blood samples were collected from each mouse by cardiac puncture and collected in anticoagulant tubes. When RNA was extracted, TAKARA PrimeScript TMRT reagent Kit with gDNA Eraser reagents, transfer blood samples to lysis containers, add lysis reagents, shake at 1500rpm for 12 minutes to fully lyse blood cells, add stratification reagents, centrifuge at 13000rpm for 7 minutes to stratify, transfer RNA-containing aqueous phase to new containers, add precipitation reagents, place on ice for 15 minutes, then centrifuge at 9000rpm for 12 minutes to precipitate RNA, wash the precipitate twice with 70% ethanol, centrifuge at 7500rpm for 7 minutes after each wash, finally dissolve RNA with 30μL of ribonuclease-free DEPC water, measure the absorbance of RNA solution at 260nm and 280nm using a UV spectrophotometer to ensure that the A260 / A280 ratio is between 1.8-2.0, and store qualified RNA samples in a -70℃ ultra-low temperature refrigerator.

[0036] Select a kit suitable for reverse transcription of the ID2 gene, operate on ice, prepare the genomic DNA removal reaction solution, add 4 μL RNA sample, 1.5 μL genomic DNA contamination removal reagent, 2.5 μL 5×gDNA Eraserbuffer to the centrifuge tube, add DEPC water to a total volume of 12 μL and mix well, then prepare the reverse transcription PCR reaction solution, take 8 μL of the genomic DNA removal reaction solution, add 3 μL 5×Primescript Buffer, 1.5 μL 5×Primescript RT Enzyme Mix, 3 μL RTPrimer Mix, add DEPC water to a total volume of 25 μL, mix gently, incubate the genomic DNA removal reaction solution at 30°C for 12 minutes, and put it into the PCR instrument, complete the reverse transcription PCR reaction according to the program of 37°C for 25 minutes and 80°C for 8 minutes to obtain the cDNA of the ID2 gene.

[0037] Add 3 μL cDNA template, 0.5 μL PCR upstream primer (10 μM), 0.5 μL PCR downstream primer (10 μM), 8 μL premixed reagent containing Taq polymerase, deoxyribonucleoside triphosphate, SYBR Green dye and reaction buffer to the PCR reaction tube on ice, and then add 8 μL sterile double distilled water to a total volume of 20 μL. Mix gently and expel bubbles by brief centrifugation if any. Seal the reaction tube and centrifuge it instantaneously to make the reaction solution evenly distributed at the bottom of the tube. Put it into the fluorescent quantitative PCR instrument and react according to the procedure of pre-denaturation at 94°C for 12 minutes, denaturation at 94°C for 3 seconds, annealing at 62°C for 25 seconds, and extension at 72°C for 12 seconds, and finally perform melting curve analysis (95°C for 20 seconds, 70°C for 1.5 minutes, and 95°C for 20 seconds).

[0038] After the reaction is completed, data processing and analysis are performed, and the original data are obtained using the instrument's own software and exported to professional data processing software. The expression level of ID2 gene was calculated by the formula. Samples at different stages of neuronal differentiation were set as the experimental group, and samples at the undifferentiated stage were set as the control group. The Ct values ​​of the target gene and the reference gene of the two groups of samples were determined respectively. △Ct and △△Ct were calculated to obtain the ratio of the expression level of ID2 gene between samples. The T.test statistical method was used to analyze the data. The results showed that Figure 3 As shown in the figure, with the advancement of neuronal differentiation, the expression level of ID2 gene showed a trend of first increasing and then decreasing, and the expression difference was statistically significant at a specific differentiation stage (P<0.05), indicating that ID2 gene may play an important regulatory role in the process of neuronal differentiation. Figure 3 In the middle, * indicates various statistical calculations relative to the control group, * indicates P < 0.05, and ** indicates P < 0.01.

[0039] Embodiment 3

[0040] A total of 60 patients with coronary heart disease and 60 healthy controls matched in age and gender were selected. Under the condition of strict compliance with aseptic operation specifications, 4 mL of venous blood samples were collected from each subject using sterile blood collection tubes, anticoagulant tubes were used for blood collection, and TAKARA PrimeScript TM RT reagent Kit with gDNA Eraser reagents, transfer the collected blood sample to the lysis container provided by the kit, add the lysis reagent, oscillate at 2500rpm for 8 minutes to promote the full lysis of blood cells, add the stratification reagent, centrifuge at 14000rpm for 9 minutes for stratification, transfer the aqueous phase containing RNA to a new container, add the precipitation reagent, place on ice for 25 minutes, then centrifuge at 11000rpm for 18 minutes to precipitate RNA, wash the precipitate with 78% ethanol 3 times, centrifuge at 9000rpm for 7 minutes after each wash, finally use 60μL of ribonuclease-free DEPC water to dissolve the precipitated RNA, use a UV spectrophotometer to measure the absorbance of the RNA solution at 260nm and 280nm wavelengths, ensure that the A260 / A280 ratio is in the range of 1.8-2.0, and store the qualified RNA samples in an ultra-low temperature refrigerator at -80℃.

[0041] Select a kit suitable for reverse transcription of the miR-592 gene, operate on ice, first prepare the reverse transcription reaction solution, add 6μL RNA sample, 4μL 5× Primescript Buffer, 1μL 5× Primescript RT Enzyme Mix, 4μL RT Primer for miR-592 into a centrifuge tube, and add DEPC water to the total volume of 20μL. Use a pipette to gently blow to mix all the components thoroughly. After briefly centrifuging the prepared reaction solution, put it into the PCR instrument and perform reverse transcription at 42°C for 50 minutes and 70°C for 12 minutes to obtain the cDNA of the miR-592 gene.

[0042] On ice, add 2.5 μL cDNA template, 0.6 μL PCR upstream primer (10 μM), 0.6 μL PCR downstream primer (10 μM), 9 μL premixed reagent containing Taq polymerase, deoxyribonucleoside triphosphate, SYBR Green dye and reaction buffer to the PCR reaction tube, then add 7.3 μL sterile double distilled water to make the total volume reach 20 μL, gently mix the reaction solution, if bubbles are generated, remove them by brief centrifugation, seal the reaction tube and perform instantaneous centrifugation to make the reaction solution evenly distributed at the bottom of the tube, then put it into the fluorescent quantitative PCR instrument, and react according to the program of pre-denaturation at 95°C for 11 minutes, then denaturation at 95°C for 3 seconds, annealing at 60°C for 22 seconds, and extension at 70°C for 15 seconds, and finally perform melting curve analysis (95°C for 18 seconds, 70°C for 1.2 minutes, and 95°C for 18 seconds).

[0043] After the reaction, data processing and analysis were performed. The original data were obtained using the instrument's own software and exported to a spreadsheet for organization. The expression level of miR-592 gene was calculated by the formula. The samples of patients with coronary heart disease were set as the experimental group, and the samples of healthy controls were set as the control group. The Ct values ​​of the target gene and the Ct values ​​of the reference gene of the two groups of samples were determined respectively, and the △Ct and △△Ct were calculated, and then the ratio of the expression level of miR-592 gene between the samples was obtained. The two groups of data were analyzed by the T.test statistical method. The results showed that Figure 3 As shown in the figure, the expression level of miR-592 gene in the blood of patients with coronary heart disease was significantly downregulated compared with that of healthy controls (P<0.01), indicating that miR-592 may be involved in the pathogenesis of coronary heart disease. Figure 3 In the middle, * indicates various statistical calculations relative to the control group, * indicates P < 0.05, and ** indicates P < 0.01.

[0044] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An efficient and accurate qPCR detection method for blood gene expression level, characterized in that: The method comprises the following specific steps: Blood sample processing and RNA extraction: Blood samples were collected under sterile conditions, and RNA was extracted using an adapter kit after lysis, stratification, precipitation, and washing. After being dissolved in an RNase-free solvent, its quality was tested by an ultraviolet spectrophotometer, and qualified samples were stored in an ultra-low temperature environment; Construction of reverse transcription reaction system: Select a reverse transcription kit according to the target gene, thaw the reagent on ice, and prepare the genomic DNA removal reaction solution and reverse transcription PCR reaction solution according to the instructions of the reverse transcription kit; Reverse transcription reaction: remove the genomic DNA reaction solution and incubate it, place it in a PCR instrument, and reverse transcribe the RNA into cDNA according to the temperature and time program recommended by the kit; Preparation and reaction of fluorescent quantitative PCR reaction system: prepare reagents according to the optimized formula, including cDNA template, upstream and downstream primers, premixed reagents containing Taq polymerase, deoxyribonucleoside triphosphates, fluorescent dyes and reaction buffer, and sterile double distilled water, prepare the reaction solution on ice, mix well and put it into the reaction tube, seal and centrifuge it, put it into the fluorescent quantitative PCR instrument, complete the pre-denaturation, multiple rounds of cycle reaction and melting curve analysis according to the preset program, and control the temperature and time of each step; Data processing and gene expression level calculation: After the reaction is completed, the original data is obtained using the instrument software, exported and sorted, and outliers are removed. The formula was used to calculate △Ct and △△Ct based on the Ct values ​​of the target gene and the reference gene, thereby obtaining the ratio of the target gene expression levels between samples and evaluating the gene expression difference.

2. According to claim 1, a highly efficient and accurate qPCR detection method for blood gene expression level, characterized in that: In the blood sample processing and RNA extraction step, 0.5-5 mL of blood sample is collected under sterile conditions, the collected blood sample is transferred to a lysis container provided by the kit, a matching lysis reagent is added, and the sample is shaken at an oscillation speed of 1000-3000 rpm for 5-15 minutes to fully lyse the blood cells, and then a stratification reagent is added, and the sample is centrifuged at a speed of 10000-15000 rpm for 5-10 minutes for stratification, and the aqueous phase containing RNA is transferred to a new container, and then a precipitation reagent is added, and the sample is placed on ice for 10-30 minutes, and centrifuged at a speed of 8000-12000 rpm for 10-20 minutes to precipitate RNA, and the precipitate is washed 2-3 times with 70%-80% ethanol, and after each washing, the sample is centrifuged at a speed of 7000-10000 rpm for 5-8 minutes, and 20-100 μL of RNase-free DEPC water is used to dissolve the precipitated RNA.

3. An efficient and accurate blood gene expression level qPCR detection method according to any one of claims 1 or 2, characterized in that: In the blood sample processing and RNA extraction steps, the absorbance of the RNA solution is measured at wavelengths of 260nm and 280nm using an ultraviolet spectrophotometer. When the A260 / A280 ratio is between 1.8-2.0, it indicates that the RNA purity is qualified. The RNA samples that have passed the quality inspection are stored in an ultra-low temperature refrigerator set at a temperature of -70°C to -80°C, and the samples are properly labeled and recorded.

4. The efficient and accurate qPCR detection method for blood gene expression level according to claim 1, characterized in that: In the step of constructing the reverse transcription reaction system, a genomic DNA removal reaction solution is prepared. Specifically, the reaction solution is operated on ice, and 1-10 μL of RNA sample, 0.5-2 μL of genomic DNA contamination removal reagent, and 1-4 μL of 5×gDNAEraser buffer are sequentially added to a centrifuge tube, and the total volume is supplemented with DEPC water to 10-20 μL. The components are evenly mixed by gently operating a pipette.

5. An efficient and accurate blood gene expression level qPCR detection method according to any one of claims 1 or 4, characterized in that: In the reverse transcription reaction system construction step, a reverse transcription PCR reaction solution is prepared. Specifically, 5-15 μL of genomic DNA removal reaction solution, 2-6 μL of 5×Primescript Buffer, 0.5-2 μL of reverse transcriptase 5×Primescript RT Enzyme Mix, and 2-6 μL of reverse transcription random primer RT Primer Mix are added in sequence on ice, and DEPC water is added to make the total volume 20-30 μL, and the components are evenly mixed by gently operating a pipette.

6. An efficient and accurate blood gene expression level qPCR detection method according to any one of claims 1 or 4, characterized in that: In the reverse transcription reaction step, the genomic DNA removal reaction solution is removed and incubated. Specifically, the prepared genomic DNA removal reaction solution is briefly centrifuged to concentrate the liquid in the tube at the bottom of the tube, the centrifuge tube is placed in an environment of 25°C-42°C for incubation for 5-15 minutes, and it is placed in a PCR instrument to perform reverse transcription at 37°C-42°C for 10-60 minutes to synthesize cDNA from RNA under the action of reverse transcriptase, and an enzyme inactivation step is performed at 70°C-85°C for 3-10 minutes to terminate the reverse transcription reaction and obtain a high-quality cDNA product.

7. The efficient and accurate qPCR detection method for blood gene expression level according to claim 1, characterized in that: In the preparation and reaction steps of the fluorescent quantitative PCR reaction system, reagents are prepared according to an optimized formula, including 1-5 μL of cDNA template, 0.2-0.8 μL of PCR upstream primer with a concentration of 10 μM, 0.2-0.8 μL of PCR downstream primer with a concentration of 10 μM, 5-15 μL of a premixed reagent containing Taq polymerase, deoxyribonucleoside triphosphate, SYBR Green and reaction buffer, and sterile double distilled water is added to make up the total volume to 15-30 μL.

8. An efficient and accurate blood gene expression level qPCR detection method according to any one of claims 1 or 7, characterized in that: In the preparation and reaction steps of the fluorescence quantitative PCR reaction system, the reagents are placed in a fluorescence quantitative PCR instrument, and pre-denaturation, multiple rounds of cycle reaction and melting curve analysis are completed according to a preset program, and the temperature and time of each step are controlled. Specifically, in the pre-denaturation stage, the temperature is maintained at 94°C-96°C for 5-15 minutes. In the cycle reaction stage, the denaturation temperature is set to 94°C-96°C for 1-5 seconds, the annealing temperature is 55°C-65°C for 10-30 seconds, the extension temperature is 70°C-75°C for 5-20 seconds, and the cycle is repeated 35-45 times. During the melting curve analysis, the temperature is maintained at 95°C for 10-30 seconds, the temperature is slowly reduced to 60°C-70°C, and then the temperature is increased to 95°C at a rate of 0.1°C-0.3°C / second.

9. The efficient and accurate qPCR detection method for blood gene expression level according to claim 1, characterized in that: In the data processing and gene expression level calculation steps, 2 -△△Ct Formula, △Ct and △△Ct are calculated based on the Ct values ​​of the target gene and the reference gene, so as to obtain the ratio of the target gene expression levels between samples. Specifically, CtA1 is set as the Ct value of the target gene of sample No. 1, CtB1 is set as the Ct value of the reference gene of sample No. 1, CtA2 is set as the Ct value of the target gene of sample No. 2, and CtB2 is set as the Ct value of the reference gene of sample No.

2. Then the ratio of the target gene expression levels of sample No. 1 and sample No. 2 is calculated as follows: △△Ct=(CtA2-CtB2)-(CtA1-CtB1)=X, then the expression level of the target gene in sample No. 2 is the 2nd level in sample No.

1. -X times.

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