Taq polymerase

MiRNA expression levels were measured by real-time PCR on a CFX96 amplifier (Bio-Rad Laboratories, California, USA). The reaction was carried out in a volume of 30 µL containing 3 µL of cDNA, 14 µL of H2O, 3 µL of 10× PCR buffer (Vector-Best), 3 µL of 4 mM deoxynucleotide triphosphate solution, 3 µL 10% BSA solution, 1 µL of Taq polymerase (Vector-Best, Novosibirsk, Russia) along with monoclonal antibodies to its active center (Clontech, California, USA), 3 µL of a mix of forward and reverse primer (5 µM) and probe (2.5 µM). The primers and the probes are Vector-Best (Novosibirsk, Russia) developments, the efficiency of the PCR being 85%–100%. Analysis of the threshold cycles generated by the qPCR was performed using the 2-∆Ct method. Statistical processing of data was carried out using STATISTICA v12.0 (StatSoft Inc., OK, USA). Two independent samples were compared using the Mann–Whitney U test.

MiRNA expression levels were measured by real-time PCR on a CFX96 amplifier (Bio-Rad Laboratories, Hercules, CA, USA). The total volume of each reaction was 30 μL and contained 3 μL of cDNA, 1× PCR buffer (Vector-Best, Novosibirsk, Russia), 0.4 mM of each dNTP (Biosan, Riga, Latvia), 1% BSA, 1U Taq polymerase (Vector-Best, Novosibirsk, Russia) premixed with 10× active center-specific monoclonal antibody (Clontech, Mountain View, USA), 0.5 units of uracil-DNA glycosylase (Vector-Best, Novosibirsk, Russia), 0.5 μM of each primer and 0.25 μM of TaqMan probe. The primers and the probes are Vector-Best developments, the efficiency of the PCR being 90–100%.

Detection was performed using Real-time PCR combined with reverse transcription reaction in a single tube. Total volume of each reaction was 30 μL. Reaction mix contained 3 μL of RNA preparation, 16.7 % trehalose, 1x RT-PCR buffer (Vector-Best, Russia), 0.4 mM of each dNTP, 1 % BSA, 100U M-MLV reverse transcriptase (Vector-Best, Russia), 1U Taq polymerase (Vector-Best, Russia) pre-mixed with active center-specific monoclonal antibody (Clontech, USA), 0.5 μM of each primer and 0.25 μM of dual-labeled probe. RT-PCR protocol: incubation at 45 °С - 30 min., heating at 95 °С - 2 min., 50 cycles of denaturation at 94 °С - 10 s, annealing and elongation: 60 °С - 20 s. Sequences of primers and the probe are listed in Additional file 2.