Quantitative PCR Analysis of Gene Expression

Quantitative PCR analysis was performed as previously reported^{6 (link)}. Total RNAs were extracted with Trizol (Invitrogen; Carlsbad, CA) and then reverse-transcribed using the HyperScript^TM Reverse Transcriptase (Geneall, Seoul, South Korea) in a PTC-200 Thermal Cycler (MJ Research, Reno, NV). The resulting cDNA (25 ng) was amplified using LaboPass^TM SYBR Green Q Master (Cosmogenetech, Seoul, South Korea) for quantitative real-time PCR. PCR experiments were performed in triplicate. Primer sequences are provided as Supplementary Table 1. Real-time PCR was performed in a CFX Connect^TM Real-Time PCR Detection System (Bio-Rad Laboratories; Hercules, CA). The expression of gene transcripts was normalized to the geomean values of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succineate dehydrogenase complex subunit A, flavoprotein (FP)(SDHA), and hypoxanthine phosphoribosyltransferase 1 (HPRT1)^{48 (link)}, and relative expression values were calculated according to the ΔΔCt method.