Reverse transcription was performed using the miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit (B532451; Sangon Biotech Co, Ltd., China) according to the manufacturer’s instructions. Thereafter, RT-PCR analysis was performed using the SYBR miRNA RT-PCR kit (B532461; Sangon Biotech Co, Ltd., China) on a Roche LightCycler 96 instrument (Roche, USA). The PCR program was as follows: 1) 95°C for 30s, 2) 40 cycles at 95°C for 5s and at 60°C for 30s, and 3) melting and cooling at 95°C for 10s, at 65°C for 60s, at 97°C for 1s, and at 37°C for 30s. Each sample was amplified in triplicate. The relative expressions of miRNAs were normalized to U6 expression. All the primers were synthesized by Sangon Biotech Co, Ltd., China. The sequences of the primers were as follows: MiR-25-3p: 5’-TCATTGCACTTGTCTCGGT-CTGA-3’; MiR-181a-5p: 5’-AACATTCAACGCTGTCGGTGAGT-3’.
Sybr mirna rt pcr kit
Manufactured by Sangon
Sourced in China
The SYBR miRNA RT-PCR kit is a laboratory tool designed for the detection and quantification of microRNA (miRNA) expression levels using real-time PCR technology. The kit includes components necessary for the reverse transcription and amplification of miRNA samples.
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2 protocols using sybr mirna rt pcr kit
1
Reverse Transcription and RT-PCR Analysis of miRNA
2
Validating Small RNA Sequencing with qRT-PCR
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To validate the initial results of small RNA sequencing, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted on 3 miRNAs (miR-320c, miR-181a-2-3p, and miR-619-5p). The RNA was reversely transcribed with the miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit (B532451; Sangon Biotech Co, Ltd., Shanghai, China) according to the manufacturer’s instructions. The cDNA was quantitated using the SYBR miRNA RT-PCR kit (B532461; Sangon Biotech Co, Ltd.) on a Roche LightCycler 96 instrument (Roche, Basel, Switzerland) under the following conditions: 30 seconds at 95°C, followed by 5 seconds at 95°C plus 30 seconds at 60°C for 40 cycles, then 10 seconds at 95°C, 60 seconds at 65°C, 1 seconds at 97°C, and 30 seconds at 37°C. The relative expression levels of miRNAs were normalized to an endogenous control (miR-423–5p).[18 (link),19 (link)] All primers were synthesized by Sangon Biotech Co, Ltd.
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