Total RNA was isolated from 50 mg of tissue, using TRIzol (Life Technologies, Grand Island, NY). RNA (1.5 µg) was reverse transcribed with Reverse Transcriptase (Promega, Madison, WI) in 20 µL reaction volume. The RT reaction was diluted 5-fold and 5 µL was amplified by real-time PCR (qPCR) in a 25 µL reaction mixture containing Master Sybr Green (Bio-Rad, Hercules, CA). Primers (Table S1) were designed to span the intron-exon borders, using Primer Express software (Applied Biosystems, Grand Island, NY). qPCR was performed on a MyiQ real-time PCR system (Bio-Rad, Hercules, CA): an initial 2 min denaturation step at 95° C, followed by 40 cycles of 15 sec each at 95° C and a 1 min annealing step at 60° C. Gene expression was quantified in replicate samples using the delta ct methodology [24], normalizing to cyclophillin expression.
