Technology for detecting micro RNA-150 relative content of T cells and reflecting individual immune state

Abstract

The invention discloses a stem-loop primer and multiple matched amplification primers for carrying out relative quantitative detection on micro RNA-150 nucleotide segments, a kit and an evaluation sheet. The kit comprises reagent A, reagent B, reagent C, reagent D, reagent E, reagent F, reagent G and reagent H, wherein the reagent A comprises 50muL of 5muM reverse transcription miR-150 and U6 stem-loop primer mixed solution; the reagent B comprises 50muL of 20U / muL M-MuLV reverse transcriptase; the reagent C comprises 5 reverse transcription buffer solutions, each reverse transcription buffer solution comprises 200muL of 50nM DTT, the dNTP concentration is 5mM (including dATP, dCTP, dGTP and dTTP), the Tris-HCl concentration is 250mM, the KCl concentration is 250mM, and the MgCl2 concentration is 20mM; the reagent D comprises 100muL of 2muM common primer; the reagent E comprises 100muL of 2muM miR-150 specific primer; the reagent F comprises 100muL of 2muM U6 specific primer; the reagent G comprises 20muL of 5U / muL Taq DNA polymerase; and the reagent H comprises 2 PCR buffer solutions, and each PCR buffer solution comprises 600nM ROX, one SYBR Green I dye, 2mM dNTPs (including dATP, dCTP, dGTP and dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4)2SO4 and 6mM MgSO4. The complete kit disclosed by the invention can carry out relative quantitative analysis on micro RNA-150 nucleotide segments of T cells of different types, and can evaluate the individual immune state.

Description

technical field 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 microRNA-150 (microRNA-150, miR-150) reflects the individual's T cell immune status. The introduction of 8 specific nucleotide sequences at the 3' end of the stem-loop primer can reverse transcribe miR-150, and amplify miR-150 by Q-PCR technology. The relative content of miR-150 was derived after comparison with the content of U6 non-coding RNA. The relative content of miR-150 can reflect the immune status of individual T cells. This method can evaluate the immune status of individual T cells within 72 hours, and can analyze different T cell types through sorting technology to achieve a more accurate evaluation effect. After optimizing the experimental conditions, a simple and accurate relative quantitative detection kit for miR-150 was const...

AI Technical Summary

Problems solved by technology

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