Total RNA Extraction and Real-Time PCR Analysis

Total RNA was extracted using Total RNA Mini kit (Frogga Bio Inc, Canada) as described [16 (link), 43 (link)]. The RNA concentration was measured by NanoDrop 2000c UV-VIS Spectrophotometer under 260 nm. Equal amount of RNA was used for reverse-transcription to obtain cDNA for real-time PCR amplification. The cDNA was diluted as template and performed in a real-time thermal cycler using a SYBR Green PCR Kit as follows: 40 cycles of denaturation at 95°C for 5 sec, annealing at 59°C for 30 sec, followed by an elongation step at 72°C for 30 sec. The Foxo3 was analyzed using U6 as an internal control for product normalization. All tests were performed in triplicate.