Hifair 2 first strand cdna synthesis kit gdna digester plus

RNA extraction and quantitative real-time PCR were performed as previously described.⁶³ (link) Total RNA was extracted from tissues or cells with TRIzol reagent (TIANGEN), and cDNA was synthesized with a Hifair II First-Strand cDNA Synthesis Kit (gDNA Digester Plus, YEASEN, China). qPCR was performed with a Hifair III One-Step RT-qPCR SYBR Green Kit (YEASEN), and triplicate samples were run on an ABI QuantStudio 5 real-time PCR system (Thermo Fisher, USA). The threshold cycle (Ct) values for each gene were normalized to those of GAPDH as an endogenous calibrator, and the 2^−ΔΔCt method was used for quantitative analysis. The primers used were synthesized by Sangon Biotech and are listed in Table S2. To confirm the expression of miR-6077, we isolated RNA with a miRcute miRNA Isolation Kit (Qiagen, Germantown, MD, USA), and the reverse transcription and qPCR were performed with a miRcute Plus miRNA First-Strand cDNA Kit and miRcute Plus miRNA qPCR Kit (SYBR Green, Qiagen), respectively. Small nuclear RNA (U6) was used as an endogenous calibrator.

RNA extraction and quantitative real-time PCR (qRT-PCR) were performed as previously described^{29 (link)}. Briefly, Total RNA was extracted from tissues or cells with TRIzol reagent (TIANGEN), and cDNA was synthesized with a Hifair II First-Strand cDNA Synthesis Kit (gDNA Digester Plus, YEASEN, China). qRT-PCR was performed with a Hifair III One-Step run on an ABI QuantStudio 5 real-time PCR system (Thermo Fisher, USA). The threshold cycle (Ct) values for each gene were normalized to those of Actin as an endogenous calibrator, and the 2^(−△△CT) method was used for quantitative analysis. The primers of each gene were synthesized by Sangon Biotech and are listed in Supplementary Table S1.