Quantification of Survivin mRNA Levels

Total RNA from control or PPRH-transfected cells for 24 h and 48 h was extracted using Trizol Reagent (Life Technologies, Madrid, Spain), following the instructions of the manufacturer. Complementary DNA was synthesized in a 20 µL reaction mixture from 1 µg of total RNA, 0.5 mM of each deoxyribonucleotide triphosphate (dNTP, Epicentre, Madison, USA), 250 ng of random hexamers (Roche, Barcelona, Spain), 10 mM dithiothreitol, 200 units of a Moloney murine leukemia virus reverse transcriptase (RT), 20 units of RNase inhibitor, and 4 µL of buffer (5×) (all three from Lucigen, Middleton, WI, USA). The reaction was incubated at 42 °C for 1 h.
The BIRC5 mRNA TaqMan probe (Hs04194392_s1; ThermoFisher Scientific, Madrid, Spain) was used to determine survivin mRNA levels and the Cyclophilin (PP1A) mRNA TaqMan probe (Hs04194521_s1, ThermoFisher Scientific, Madrid, Spain) was used as the endogenous control. The reaction was conducted in 20 μL containing 1xTaqMan Universal PCR Mastermix (Applied Biosystems, Madrid, Spain), 0.5xTaqMan probe, and 3 μL of cDNA. PCR cycling conditions were 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Barcelona, Spain). The quantification was performed using the ΔΔCt method, where Ct is the threshold cycle that corresponds to the cycle when the amount of amplified mRNA reaches the fluorescence threshold.