Quantitative real-time PCR (qPCR). One micro gram of RNA extracted from cardiac myocytes and cDNA was prepared using Superscript II (Invitrogen, Paisley, UK) and random hexamers (Promega, UK). qPCR was carried out using Platinum SYBR Green (Invitrogen, Paisley, UK) on the DNA Engine Opticon system (MJ Research, Waltham, MA). HPRT, Actin and GAPDH were used together as normalising Telaprevir
and for each experiment both target and normalising gene PCR efficiency was firstly determined to ensure normalizing genes were acceptable. To test primer efficiency, qPCR was carried out on a 2-fold dilution series from a pooled set of cDNA and the threshold Ct value was plotted against the log cDNA dilution. Efficiency was then calculated using the equation m = (?1/log E), where m is the slope of the line and E is the efficiency and primer pairs were used only if the PCR efficiency of the normalising and control genes
were found to be within 10% of each other. Expression changes were claculated using the 2?ΔΔCt method and expressed as fold change over control . Specific primers were designed with the aid of CloneWorks and the Ensembl database and are shown below.