2.9. RNA extraction, reverse transcription and qPCR

The RNA extraction was performed as previously described. 29 One (for H295R) or 2 (for SW13) micrograms of total RNA were reverse‐transcribed in a final volume of 50 μL using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific); cDNA was diluted 1:2 in DNAse and RNAse free water. Primer sequences are shown in Table 1. PCR reactions were performed in the QuantStudio^Tm 3 Real Time PCR System (Thermo Fisher Scientific) using 0.3 (for H295R) or 0.6 (for SW13) μmol/L of each primer. PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific) with the dissociation protocol was used for gene amplification; negative controls contained water instead of first‐strand cDNA. Each sample was normalized to its 18S rRNA (18S) content. Final results were expressed as n‐fold differences relative to a calibrator and calculated using the ΔΔCt method.

Primers oligonucleotide sequences. The indicated primers oligonucleotide sequences were used for the amplification of following genes: SIRT1: sirtuin 1; ESR1: oestrogen receptor alpha; CCND1: cyclin D1; IGF1R: insulin growth factor receptor 1; 18S: 18S ribosomal RNA