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A triple RT-qPCR detection method and kit for u6, mir 92a and miR 21

A detection kit and the technology of the kit, which are applied in the fields of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of time-consuming and labor-intensive, inaccurate quantification, and excessive amplification.

Active Publication Date: 2020-11-20
东莞微量精准检测研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

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  • A triple RT-qPCR detection method and kit for u6, mir 92a and miR 21
  • A triple RT-qPCR detection method and kit for u6, mir 92a and miR 21
  • A triple RT-qPCR detection method and kit for u6, mir 92a and miR 21

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 A detection kit for triple RT-qPCR of U6, miR 92a and miR 21

[0077] The kit includes: U6, miR 92a and miR 21 triple RT-qPCR reverse transcription stem-loop primer set.

[0078] RT stem-loop primer sets include:

[0079] Primer stem-loop primer 21: 5'-CTC AAC TGC TCT GCT CCA GTC GCG GAT TCAGTT GAG TCA ACA T-3';

[0080] Primer stem-loop primer 92a: 5'-GTC GTA TCC AGT CGT CGC TCC GAC TCG CGCTGG ATA CGA CAC AGG CCG-3';

[0081] Primer stem-loop primer U6: 5'-GTC GTA TCC AGC TCT GCT CCA GTC GCG GATTCT GGA TAC GAC AAA ATA-3'.

Embodiment 2

[0082] Example 2 A detection kit for triple RT-qPCR of U6, miR 92a and miR 21

[0083] The kit includes: (1) the reverse transcription stem-loop primer set for the triple RT-qPCR of U6, miR 92a and miR 21 described in Example 1; (2) the triple RT-qPCR primer set for U6, miR 92a and miR 21 Quantitative detection primer set and quantitative detection probe set.

[0084] Quantitative detection primer sets include:

[0085] Quantitative detection primer set 1 and quantitative detection probe set 1;

[0086] And quantitative detection primer set 2 and quantitative detection probe set 2.

[0087] Quantitative Detection Primer Set 1 includes:

[0088] Quantitative PCR upstream primer m2102: 5'-CCC CGG TAG CTT ATC AGA CTG-3'; quantitative PCR downstream primer m2103: 5'-CTC AAC TGC TCT GCT CCA GT-3'; quantitative PCR upstream primer U602: 5'-CTC GCT TCGGCA GCA CA-3'; Quantitative PCR downstream primer U603: 5'-AAC GCT TCA CGA ATT TGC GT-3';

[0089] Quantitative detection probe s...

Embodiment 3

[0097] Example 3 A detection kit for triple RT-qPCR of U6, miR 92a and miR 21

[0098] The kit includes:

[0099] (1) the reverse transcription stem-loop primer set in embodiment 1;

[0100] (2) quantitative detection primer set and quantitative detection probe set in embodiment 2;

[0101] (2) Enzyme system: MMLV DNA polymerase, Tfl DNA polymerase and Stoffel fragment;

[0102] (3) PCR reagent: Tris-H at pH 8.5 2 SO 4 , 3-(N-morpholino)propanesulfonic acid, pH 7.9, sodium citrate, (NH 4 ) 2 SO 4 , MgSO 4 , polyoxyethylene lauryl ether, acetylated bovine serum albumin and dNTPs.

[0103] (4) Quantitative standard: quantitative standard 1 and quantitative standard 2.

[0104] According to the sequence of miRNA and stem-loop primers, miR 21 plasmid, miR92a plasmid and U6 plasmid were synthesized in Shanghai Jierui Bioengineering Co., Ltd. The miR 21 plasmid contains the nucleotide sequence shown in SEQ ID NO: 14 (5'-CTC AAC TGC TCT GCT CCA GTCGCG GAT TCA GTT GAG TCA AC...

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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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2537/143C12Q2521/107C12Q2545/113
Inventor 张新彭春梅张晓玮邓可基乐小炎张嘉李家导陈观芝林敏深林若琳石壮壮罗园香莫静嫣李海茵王星王法吴彩虹
Owner 东莞微量精准检测研究院有限公司
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