Quantification of Apoptotic Genes by qRT-PCR

Total RNA was isolated using TRIzol agent (Invitrogen, Carlsbad, CA, USA), and each RNA sample was reverse-transcribed to complementary DNA (cDNA) by PrimeScript™ RT Reagent Kit (Takara, Dalian, Liaoning Province, China). cDNA was used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The sets of primer pairs of apoptotic regulating genes are listed in Table 1 [29 (link)]. For qRT-PCR reactions, the 25 μL reaction mixture included 2 μL cDNA, 12.5 μL SYBR Premix Ex TaqTM Ⅱ (Takara), 1.0 μL of forward and 1.0 μL of reverse primer and 8.5 μL RNAase-free water (Takara). Reaction conditions were 95 °C for 3 min followed by 44 cycles of 95 °C for 10 s, the specific melting temperature (Tm) of a primer pair for 30 s, and then 95 °C for 10 s, and 72 °C for 10 s, using a Bio-Rad IQ5 Thermal Cycler (Bio-Rad). β-actin was selected as a reference gene. The expression fold changes were calculated using the 2^−ΔΔCT method [30 (link)].