RNA Extraction and qPCR Analysis

Total RNAs from tissues were extracted using Tri-RNA reagent from Sigma (St Luis, MO) according to the manufacturer’s instruction and 1 microG of total RNA was reverse transcribed into cDNAs using the first-strand synthesis kit (Roche, Tucson, AZ, USA). Real-time quantitative PCR analyses were performed according to a previously published procedure [47 (link)]. Triplicate CT values were analyzed in Microsoft Excel using the comparative Ct(DDCt) method as described by the manufacturer (Applied Biosystems, Foster City, CA, USA). The amount of target (ddCt) was obtained by normalization to an endogenous reference (Gapdh for mice) and relative to a calibrator. All TaqMan primers and probes were purchased from Applied Biosystems Inc.