Superscript vilo enzyme

After 72 hours, the tissue was collected and total RNA was extracted with RNA-Bee solution (Tel-Test Inc., Friendswood, TX) according to the manufacturer’s instructions. RT was performed using the SuperScript VILO enzyme (Invitrogen, Grand Island, NY). The reaction protocol consisted of annealing at 25 °C for 10 min, followed by one cycle at 42 °C for 60 min and terminated by incubation at 85 °C for 5 min. The cDNA was then used for real time PCR in the LightCycler 480 instrument using the SYBR Green I Master Mix (Roche, Indianapolis, IN) and primers specific for the rat SCN5A gene (Supplementary Table 2). The standard protocol used was the following: 5 min at 95 °C, followed by 45 cycles of 10 s at 95 °C, 10 s at 60 °C and 10 s at 72 °C. Melting curves generated with a dissociation protocol were used to ensure the specificity of primers for a single product for each reaction. The expression of SCN5A was normalized to β-actin and the level of knockdown was calculated using the ΔΔCt method. Levels of RNA were measured in triplicate from each knockdown experiment on tissues from n = 5 rats.