Sybr green reagent qpcrmix hs sybr highrox

Reaction products after gDNA amplification with all primer pairs used in the subsequent qPCR analysis of gene expression were cloned into a pGEM-T Easy vector (Promega, USA) for determination of primer efficiencies with the help of a standard curve method performed on an AB StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Carlsbad, CA, USA) using SYBR Green reagent qPCRmix-HS SYBR + HighROX (Evrogen, Moscow, Russia) and analyzed by means of StepOne Software v.2.3 (R2 > 0.991 for all primer pairs and efficiencies 70–90%). Primer efficiencies used in the further estimation of fold difference of target sequences expression with delta CT method were calculated as follows (1): $E={10}^{\left(-\frac{1}{a}\right)},$
where E is a primer pair efficiency and a is the slope obtained from the standard curve.

Reverse transcription was performed in the reaction volume of 10 µL using MMLV RT kit (Evrogen, Moscow, Russia), 2 µg of total RNA was reverse transcribed using random hexamer primer and 100 units of MMLV RT at 42 °C for 60 min.
The comparative C_T experiment was performed on AB StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Carlsbad, CA, USA) using SYBR Green reagent qPCRmix-HS SYBR + HighROX (Evrogen, Moscow, Russia) with the reaction set up according to the manufacturer’s instructions. The reactions were performed in triplicates and included negative controls in which RNA was added instead of the template. The run protocol consisted of 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s, with fluorescence intensity measurement performed after the elongation stage. The specificity of PCR products was confirmed using a melting curve analysis. The expression of target sequences in alpha-amanitin treated and non-treated cells was normalized to that of the 18S rRNA. Analysis of the fold difference in the expression of the regions of interest were performed with StepOne Software v2.3 (Thermo Fisher Scientific, USA). The primers for the regions of interest, 18S rRNA, GAPDH, and TBP are given in the Appendix A (Table A1).