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Relative quantification kit for detecting ribonucleic acid of Thl/Th2 cell factors

A technology of kits and reagents, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long analysis time, high throughput, and high requirements for specimens, and achieve reliable detection technology and shorten The effect of experiment time and optimized reaction conditions

Inactive Publication Date: 2011-08-17
余震 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the deficiencies in the existing immunodiagnostic technology and molecular diagnostic technology in the evaluation technology of individual immune status, including defects such as long time-consuming analysis, incapable of high throughput, and high requirements for specimens

Method used

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  • Relative quantification kit for detecting ribonucleic acid of Thl/Th2 cell factors
  • Relative quantification kit for detecting ribonucleic acid of Thl/Th2 cell factors
  • Relative quantification kit for detecting ribonucleic acid of Thl/Th2 cell factors

Examples

Experimental program
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Embodiment 1

[0031] First, after wetting the injection syringe with heparin, take about 3ml of venous blood routinely, and turn the syringe to mix the blood evenly. Add whole blood to the culture bottle (5ml of 1640 culture solution containing 20% ​​serum, 5mg of PHA, pH 7.2) in the ultra-clean workbench, add 0.5ml of whole blood (13-15 drops of No. 7 needle), and tightly cover the rubber stopper , shake gently. Place the culture flask in a constant temperature incubator at 37°C for 16 hours. Collect uncultured and cultured blood cells in centrifuge tubes, and centrifuge at 1000rpm for 5min after balancing, remove the supernatant and leave 1ml, and mix thoroughly. Add 25 μL of T cell magnetic beads after mixing and invert repeatedly 2-3 times to disperse the magnetic beads. Gently rotate for 3 minutes at room temperature to allow the beads to attach to the T cells (do not exceed 4 minutes). Mixing can be done using an inversion device or by hand. Place on the magnetic stand for 3 minut...

Embodiment 2

[0033] A reverse transcription operation procedure for detection of target mRNA matched with the method. 20 μL system contains 2 μL mRNA (0.5pg-1μg), primer mixture reagent A 2 μL, reverse transcriptase reagent B 2 μL and reverse transcription buffer reagent C 4 μL (final concentration of dNTP is 1 mM, primer is 500 nM, M-MuLV reverse transcription Enzyme concentration is 40U, Tris-HCl concentration is 50mM, KCl concentration is 50mM, MgCl 2 Concentration is 4mM), add water 10μL. The reverse transcription program was placed on ice for 5 minutes, 30°C for 30 minutes, and 95°C for 5 minutes. After the reverse transcription is completed, 180 μL of water is added to the test tube to make a 10-fold dilution.

Embodiment 3

[0035] A Q-PCR method for detecting mRNA of Th1 / Th2-related cytokines, as well as an operation process and a reaction program. Each item requires the detection of 2 repeated tubes, a total of 14 reaction tubes, and the reaction system of each reaction tube can be 10μL-50μL. In the 10 μL Q-PCR system, first take 14 μL of diluted cDNA and add it to a blank tube, add 70 μL of specific primers, add 56 μL of water to mix, take out 10 μL each time and add them to tubes 1-7 (a total of 2 tubes). The final concentrations in each tube were: primer 200nM, 300nM ROX, Green I dye, 1mM dNTPs, 40mM Tris-HCl, 40mM KCl, 20mM (NH 4 ) 2 SO 4 , 3mM MgSO 4 , Taq enzyme 0.5U. Apply the film and test on the ABI 7500. The procedures for cDNA amplification and quantification are as follows: 3 minutes of pre-denaturation at 95°C; 40 cycles of denaturation at 95°C for 5 sec and annealing and extension at 62°C for 35 sec (fluorescence value is collected at this step). The Ct value of each PCR tu...

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Abstract

The invention discloses a reverse transcription primer which can be applied to relative quantification detection of relevant cell factors of a Thl / Th2 cell, a plurality of associatively amplified gene specific primers and a kit. The kit comprises a reagent A: 50muL of 5muM reverse transcription primer, a reagent B: 50muL of 20U / muL M-MuLV reverse transcriptase, a reagent C: 200muL of 5*reverse transcription buffer solution containing 5mM of dNTP (deoxy-ribonucleoside triphosphate) including dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate)], 250mM of Tris-HCl, 250mM of KCl, 20mM of MgCl2 and 50nM of DTT (dithiothreitol), a reagent D: 2*PCR (Polymerase Chain Reaction) buffer solution, wherein 1mL of buffer solution contains 100 U of taq enzyme, 600nM of ROX (roxithromycin), Green I dye, 2mM of dNTPs (including dATP, dCTP, dGTP and dTTP), 80mM of Tris-HCl, 80mM of KCl, 40mM of (NH4)2SO4 and 6mM of MgSO4, and 24 primer-containing PCR 8 cascade reaction tubes. The entire set of kit provided by the invention can be applied to relative quantification of the cell factors of the Thl / Th2 cell; moreover, relative quantification analysis can be performed on mRNA (messenger ribonucleic acid) fragments of six types of cell factors of different types of T cells, and the immune state of an individual can be estimated according to a result.

Description

technical field [0001] The invention relates to biotechnology, is a molecular biology method based on polymerase chain reaction (PCR), in particular relates to fluorescent quantitative polymerase chain reaction (Q-PCR). The relative content of mRNA of multiple immune-related molecules is used to reflect the immune status of individual T cells. Introducing 11 specific nucleotide sequences at the 3' end of the primer for synthesizing cDNA can reverse transcribe mRNA, and a high-quality nucleotide sequence at the 5' end as a label (also the position of the primer) and the opposite gene Specific primers were used for Q-PCR to amplify and relative quantify various mRNAs. After comparing with the content of GAPDH, the relative content of various mRNAs was obtained. The relative content of IL-2, INF-γ, IL-12, IL-4, IL-10, and TGF-b1 mRNA can reflect the immune status of individual T cells, especially the balance of Th1 / Th2 cells. This method can relatively quantify the expression ...

Claims

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Application Information

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IPC IPC(8): C12Q1/32C12N15/11C12Q1/68
Inventor 余震陈必成洪炜龙杨丽红王斯璐白永恒潘晓东
Owner 余震
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