2.2. RNA Isolation, cDNA Synthesis, and Real-Time PCR

RNA was isolated using TRIzol™ Reagent (Invitrogen™) according to the manufacturer's protocol. Glycogen (Thermo Scientific™, USA) was used as an RNA coprecipitant. RNA integrity was checked by running agarose gel electrophoresis. RNA concentration and purity were assessed using an Agilent-8453 spectrophotometer (Agilent Technologies, USA) at wavelengths of 260 and 280 nm. Reverse transcription was performed by using the RT-M-MuLV-RH kit (BiolabMix, Russia) according to the manufacturer's protocol. RNA at concentration of 0.8 μg was used for reverse transcription reaction. cDNA was used in real-time PCR to measure the level of AR mRNA by adding a BioMaster HS-qPCR SYBR Blue (2x) (BiolabMix) reaction mix, followed by applying the CFX96™ Detection System (Bio-Rad Laboratories, USA). SYMPK and POLR2A were used as reference genes. The following specific primers were used in this study: AR 5′-CCTGGCTTCCGCAACTTACAC-3′, 5′-GGACTTGTGCATGCGGTACTCA-3′; SYMPK 5′-GCTGGAGAAGAAAGAGGTG-3′, 5′-ACAGGTTGGTGGCTTTGATG-3′; and POLR2A 5′-GCATGGCAGAGGAGTTTCGGCT-3′, 5′-ATTTCCCCGGGATGCGCAATGG-3′.

The optimal concentration for each primer was 300 nM.

Each PCR reaction was performed by using 0.3 μl cDNA, at final volume 20 μl, under the following conditions [12]: initial denaturation for 5 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C, annealing for 20 s at 62°C, elongation, and fluorescence data processing for 30 s at 72°C. Melting profiles were used to assess PCR specificity. In each experiment, one plate contained samples of analyzed cDNA with a primer for AR gene and the reference genes (3 replicates per sample). The relative gene expression level was assessed based on threshold cycle (Ct) values considering PCR efficacy (E) for both the analyzed and reference genes.