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.
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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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