Cardiac Gene Expression Profiling

The alteration of gene expression levels in cardiac tissues, including peroxisome proliferator-activated receptor gamma co-activator 1a (PGC-1α) [2 (link)], mitochondrial transcription factor (mtTFA) [2 (link)], nuclear respiratory factor 1 (NRF1) [2 (link)], C/EBP homologous protein (CHOP) [19 (link)], activating transcription factor 4 (ATF4) [19 (link)], Bcl-2 [20 (link)], and Bax [20 (link)], was measured using a quantitative real-time polymerase chain reaction (qRT-PCR). Trizol reagent (Invitrogen, Cergy Pontoise, France) was used according to the manufacturer’s protocol for the extraction of total RNA from the frozen left ventricles of cardiac tissues. cDNA reverse transcription kits (Applied Bio-systems, CA, USA) were used to synthesize cDNA from isolated total RNA. qRT-PCR was performed using cDNA with the SYBR Primix Ex Taq (TaKaRa, Kyoto, Japan) on a CFX96TM real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The reaction volume was 20 mL and the final working concentrations of all primers were 200 nmol/L. The obtained qPCR data were analyzed through the ΔΔCT method using a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using Bio-Rad CFX Manager Ver. 2.1 software (Bio-Rad Laboratories, Hercules, CA, USA). Product specificity was confirmed by melting or dissociation curve analysis. Primer sequences are presented in Table 1.