In brief, 50 mg of tissue from each testicle was homogenized with 1 ml of TRIZOL (Invitrogen Co., USA) for 5 min at 25°C. The nucleic acids were then extracted by centrifugation according to the supplier's recommendation. The RNA concentration was evaluated by NanoDrop using the A260/A280 ratio. The quality of the extracted RNA was confirmed by 1% agarose gel electrophoresis and observation of the 28S and 18S ribosomal RNA bands. A DNase treatment was performed (Fermentas, 1 μg RNA, 0.5 μl RNA inhibitor, 1 μl DNase1, 2 μl buffer, and 15.5 μl sterile water), and the RNAs were transformed into cDNA using a reverse transcription kit according to the manufacturer's protocol (Takara). Finally, a quantitative SYBR® Green RT-qPCR was performed using a thermal cycler (Applied Biosystems; ABi) in a total volume of 10 μl containing 2.8 μl H2O, 1 μl cDNA, 1 μl specific primers, 0.2 μl ROX, and 5 μl SYBR® Green. The CT comparison method (ΔΔCT) was used, and normalization to the GAPDH gene was performed. The primers were designed by Beacon Designer 7 and are shown in Table 1.

List of primers used for real-time RT-PCR analysis.

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