Quantitative PCR for Gene Expression Analysis

The RNA was isolated and purified from frozen rat tissues using PureLink^® Micro-to Midi Total RNA Purification System from Invitrogen (Carlsbad, CA, USA) and following the manufacturer’s protocol. Equal quantities (1.0 µg) of total RNA from each tissue were converted to first-strand cDNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA) for RT-PCR. First-strand cDNA was used for qRT-PCR. Primers for qRT-PCR were intron-spanning (Supplemental Table S1). Quantitative PCR and melt-curve analyses were performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and an iCycler machine. Relative quantities of expression of the genes of interest in different samples were calculated by the comparative Ct (threshold cycle) value method [39 (link),79 (link),80 (link)].