RNA Extraction and Quantitative PCR

Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland) was used to extract total RNA. The procedure was performed according to the manufacturer’s protocol. RNA concentration and purity were assessed using a microplate reader Infinite M200Pro (Tecan) at 260 nm and with a 260/280 nm ratio, respectively. Complementary DNA (cDNA) was synthesized using 1000 ng of total RNA, 300 ng of random primers, and 1 μL of SuperScript^® II Reverse Transcriptase (Invitrogen Thermo Fisher Scientific, Carlsbad, CA, USA). To assess transcript levels of target genes, a quantitative real-time polymerase chain reaction was performed using the Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Mortlake, VI, Australia). cDNA was amplified using 200 nM forward primer, 200 nM reverse primer, KAPA SYBR FAST qPCR Kit Universal 2X qPCR Master Mix (Sigma-Aldrich), and 25 ng of cDNA with annealing temperature 56 °C. The sequences of forward and reverse primers for NOXA and MCL-1 were shown elsewhere [45 (link),46 (link)]. To calculate the relative level of each transcript versus a reference gene RPS17 (primers: 5′-AAT CTC CTG ATC CAA GGC TG-3′ and 5′-CAA GAT AGC AGG TTA TGT CAC G-3′), a mathematical model with an efficiency correction was used [47 (link)]. (Supplementary Equation (S1))