U6, miR 92a and miR 21 ternary RT-qPCR detecting method and kit

Abstract

The invention belongs to the field of biological detection, and particularly relates to a detecting method for carrying out single-tube ternary reverse transcription on three kinds of miRNA including U6, miR 92a and miR 21, a detecting method for carrying out single-tube double quantification respectively after reverse transcription and a kit. The kit comprises a reverse transcription stem-loop primer group, a quantitative detecting primer group, a quantitative detecting probe group, an enzyme system and a PCR reaction reagent; reverse transcription of U6, miR 92a and miR 21 can be carried out in the same reaction tube; meanwhile, by high-sensitivity and high-specificity quantitative detecting primer sequences and quantitative detecting probes and PCR reaction procedures and conditions which are matched with the high-sensitivity and high-specificity quantitative detecting primer sequences and quantitative detecting probes, miR 21 and U6 as well as miR 92a and U6 can be subjected to single-tube double quantification, operation errors during single-tube single quantification in the prior art are avoided, and quantification is simple and accurate. In addition, a double-polymerase amplification for Stoffel fragments and Tfl DNA polymerase is introduced, while detection specificity is improved, lots of templates can be tolerated, and detection sensitivity and result stability are improved.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a single-tube triple reverse transcription detection method for U6, miR92a and miR 21 three miRNAs, a single-tube double quantitative detection method and a kit after reverse transcription. Background technique [0002] MicroRNA (miRNA) is a kind of non-coding single-stranded small molecule RNA with a length of 20-24 nucleotides, located in the non-coding region of the genome, and has high conservation, timing and tissue specificity. miRNA plays an important role in various physiological and pathological processes such as development, cell proliferation, apoptosis, lipid metabolism, hormone secretion and tumorigenesis. [0003] miR 21 is one of the human miRNAs discovered earlier, and it exists widely in various organisms and mammalian tissues. A large number of studies have shown that miR 21 can be highly expressed in various tumors as a proto-oncogene and participate in the p...

AI Technical Summary

Problems solved by technology

If it is necessary to quantify multiple miRNAs in a sample, the reverse transcription needs to be carried out separately, and the steps are cumbersome. From reverse transcription to quantitative PCR, the workload is heavy

In addition, when miRNA is quantified, an internal reference gene is often required. When quantifying with a single tube and single weight, the reaction conditions are unstable, there are errors in adding samples, the quantification is inaccurate, and it is time-consuming and labor-intensive, which is cumbersome.

In addition, the stem-loop primer and the quantitative primer are in the same system, and the primer dimer is serious, which is easy to cause excessive amplification.

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