Quantitative Analysis of Cardiac mRNA

Total RNA from whole hearts was extracted with the mini-RNA kit according to manufacturer's instructions (Qiagen). The reverse transcription reaction (RT) was performed using MMLV superscript reverse transcriptase (Invitrogen) and random hexamers (Operon) as described elsewhere [47] (link). The final RT reaction was diluted 10-fold in nuclease free water (Sigma). All Taqman qPCR reactions were performed as described previously [48] (link) using the Chromo4 Real-Time PCR Detector (BioRad). Estimation of mRNA copy number was determined in triplicate for each RNA sample by comparison to the geometric mean of three endogenous housekeeping genes (Primer Design) as described [49] (link). Primer and probe sets for genes of interest were purchased from Primer Design or ABI.