Biomaster hs qpcr sybr blue mix

After in vitro transfection of B16 and RLS₄₀ cells as described in Section 2.2, total RNA was extracted from tumor cells at time points of 24 and 72 h using TRIzol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. The level of miRNAs was measured using stem-loop qPCR technology [37 ,38 (link)]. cDNA synthesis was carried out using MuML-V reverse transcriptase (Biolabmix, Novosibirsk, Russia) according to the manufacturer’s protocol. The RT and PCR primers used in the study are listed in Table S1. PCR amplification was carried out using BioMaster HS-qPCR SYBR Blue mix (Biolabmix, Novosibirsk, Russia) according to the manufacturer’s protocol. The obtained qPCR data were analyzed by standard Bio-Rad iQ5 v.2.0 software. For each sample, the threshold cycle (Ct) was determined. Quantitative assessment of the level of transcript representation, relative miRNA and mRNA expression was performed by comparing the Ct values for miRNA and mRNA with HPRT1 and GAPDH mRNAs used as references.