Mirna rt qpcr starter kit

Total RNA was isolated from tissues and cells using Trizol reagent (Invitrogen, USA) based on the manufacturer’s recommendations. RNA quality and concentration were measured using NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). For mRNA, All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Transgene, China) was used for RT-qPCR, while for miRNA, the miRNA RT-qPCR Starter kit (RiboBio, China) was used. The CMTM7 and GAPDH primers were synthesized by Genewiz (Tianjin, China), and miR-182-5p (ID: 002334) and U6 (ID: 001093) probes were purchased from Thermo Fisher Scientific (USA). All specific sequences are listed in Additional file 1: Table S2.

The expression of hub miRNAs that could interact with hub mRNAs and circRNAs was detected in the samples by real-time quantitative PCR (RT-qPCR). Total RNA was purified from those samples. In the case of miRNA PCR, the miRNA RT-qPCR Starter Kit (RiboBio, Guangzhou, China) was used to reverse-transcribe miRNAs following the manufacturer's instructions. Total miRNAs were reverse-transcribed to cDNAs. Then, PCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) with miDETECT A Track miRNA primers (RiboBio, Guangzhou, China) in QuantStudio 5 Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA). Relative miRNA expression was normalized to U6. In the case of circRNA PCR, the reaction was carried out using the RiboBio circRNA qRT-PCR Starter Kit (RiboBio, Guangzhou, China) and SYBR Green Master Mix (Vazyme, Nanjing, China). Relative circRNA expression was normalized to GAPDH expression. Significant miRNAs and circRNAs were identified depending on previous reports and predicted results.