Myiq5 icycler

Total mRNA was isolated from cardiac tissues using RNA‐binding columns (RNaqueous‐4‐PCR; Ambion, Austin, TX), according to the manufacturer's protocols. RNA was immediately reverse transcribed into cDNA using a kit employing random primers (iScript; Bio‐Rad, Hercules, CA), according to the manufacturer's directions. Real‐time reaction mixtures contained 11 μL of H₂O, 12 μL of IQ SYBR Green Supermix (Bio‐Rad), 1 μL of primers, and 1 μL of cDNA template (25 μL total). Sense and antisense primers were chosen from the RTPrimerDB public primer database for mouse. Reactions were aliquoted into 96‐well plates, plates were sealed, centrifuged at 500g for 60 seconds, and then samples were amplified for 40 cycles of 10 seconds at 95°C, 30 seconds at 55°C, and 10 seconds at 72°C. Real‐time PCR was performed in a MyIQ5 iCycler (Bio‐Rad). All samples were measured in triplicates and normalized to GAPDH controls run on the same plate for each mouse cDNA sample that was tested. Gene expression levels were calculated by using (2^{Ct (gene)}/2^{Ct (GAPDH)})×1000 to standardize to GAPDH and expressed as relative transcript numbers. Positive gene expression was additionally visualized by conventional PCR and ethidium bromide stained agarose gel electrophoresis using appropriate primer sets.