Total RNA was isolated using the TRIzol extraction method according to manufacturer’s recommendations, then subsequently treated with DNase. One to 2 micrograms of DNase treated-RNA were used to synthesize first strand cDNA after priming with random hexamers (Invitrogen, Waltham, MA, USA) using MuLV reverse transcriptase (Life Technologies, Grand Island, NY, USA) or using a cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time-PCR (qRT-PCR) was carried out as described previously (28 (link)) using SYBR Green master mix (Bio-Rad, Hercules, CA, USA) and transcript-specific primers that have been described previously (25 (link),27 (link),29 (link)–32 (link)). Levels of the mRNA of interest were normalized to the levels of the mRNA for 36B4 (acidic ribosomal phosphoprotein P0) in the same sample. Relative expression was calculated as the difference (ΔCt) between the Ct (threshold cycle) values of the target gene and of 36B4, and expressed as 2−ΔCt.