Sybr green 1 testing system

Total RNA of rat caput, corpus and cauda epididymal tissues was extracted using RNAprep pure Tissue Kit (TIANGEN BIOTECH, Beijing, China). Reverse transcription was performed according to the protocol of the PrimeScript^TM RT reagent Kit (Takara, Tokyo, Japan). qPCR was performed according to the manufacturer protocols of SYBR Green I testing system (TOYOBO, Osaka, Japan) on a LightCycler 480 instrument (Roche, Basel, Switzerland). Specific primer sequences were as follow: CBS forward primer, 5′-TGAGCAGATCCAATACCGCAA-3′, CBS reverse primer, 5′-ACTCTATTTCCGGGTCTGCTC-3′; CSE forward primer, 5′-TTCCAGCACTTTGCCACTCA-3′, CSE reverse primer, 5′-CGAAGGTCAAACCGAGGACT-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward primer, 5′-GGAGTCAACGGATTTGGTCGTA-3′, GAPDH reverse primer, 5′-CTTGATTTTGGAGGGATCTCGC-3′. The PCR conditions consisted of 40 cycles of denaturation at 95°C for 5 s, annealing at 58°C for 10 s, and polymerization at 72°C for 30 s. The relative quantities of mRNAs were normalized using GAPDH as the internal control gene. The amplification efficiency of CBS/CSE primer is consistent with the efficiency of GAPDH primer and 2^-ΔΔCT method is used for the data analysis.