First strand synthesis kit

Total RNA was isolated from cells using TRIzol reagent (Sigma) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed into cDNAs using the First-Strand Synthesis Kit (Roche, Indianapolis, IN, USA). The abundance of transcripts in the cDNA samples was measured by real-time PCR with specific probes as instructed from the provider and previously described [65 (link), 66 (link)]. All probes for real-time PCR were purchased from the Applied Biosystems.

RNA purification and quantification was performed as described previously [33 (link)]. In brief, tissue powder was homogenized in Tri reagent (Sigma) with the Mini Bead-Beater (Biospec products, Bartlesville, OK, USA). To remove genomic DNA, RNA was precipitated with 2 M LiCl for at least 30 min at −20 °C. RNA integrity was checked by denaturing gel electrophoresis. RNA concentration was determined with a NanoDrop-ND-1000 spectrophotometer at 260 nm (Isogen Life Sciences, Wilmington, DE, USA). 400 ng of total RNA was transcribed using a first-strand synthesis kit (Roche). Quantitative PCR was performed in a Lightcycler 480 (Roche), using Lightcycler 480® SYBRgreen mastermix (Roche) and the following settings: denaturation: 30 s at 95 °C; annealing 30 s at 60 °C; elongation 30 s at 72 °C; 45 cycles; and a final elongation step for 5 min at 72 °C. If reverse transcriptase was omitted, no product was formed. Primary fluorescence data were quantified as described [33 (link)] and expressed relative to that of 18S rRNA. Primer sequences are given in Additional file 1: Table S1.