Biorad cfx opus 96 real time system

To analyze gene expression, total RNA was isolated with an Aurum™ Total RNA Mini Kit (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. RNA was quantified using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc, Wilmington, DE, USA). cDNA was obtained via the reverse transcription of RNA using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. For qRT-PCR, cDNA was amplified with specific primers, using qPCRmix-HS SYBR (Evrogen, Moscow, Russia) in the BioRad CFX Opus-96 real-time system (BioRad, Hercules, CA, USA) according to the kit’s enclosed protocol. The volume of RT and PCR reactions was 20 μL. Expression of target genes was normalized to GAPDH or actin gene. Primers and reaction conditions are presented in Table 1.

To analyze gene expression, total RNA was isolated with Aurum™ Total RNA Mini Kit (Bio-Rad Laboratories, USA) according to the manufacturer’s instructions. RNA was quantified in the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA). cDNA was obtained by reverse transcription of RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. For qRT-PCR cDNA was amplified with specific primers, using qPCRmix-HS SYBR (Evrogen, Russia) in the Bio-Rad CFX Opus-96 real time system (Bio-Rad Laboratories, USA), according to the kit’s enclosed protocol. Expression of target genes was normalized to GAPDH gene. Expression in MSC at early (6p) passage was applied as control. Normalization was calculated using the ΔΔCt method. All amplifications were performed in 3 technical replicas. All experiments were performed as three biological repeats.