Gene Expression Analysis in Testes

After removal of the tunica albuginea from testes tissue, RNA isolation using TRIzol reagent according to manufacturer's protocol (Life Technologies) was performed. The concentration and purity of isolated RNA was measured using NanoDrop (Peqlab). After DNaseI treatment of RNA, cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, now Thermo Fisher Scientific). qRT-PCR was performed using Maxima SYBR Green qPCR Master Mix (Life Technologies) on ViiA 7 Real Time PCR System (Applied Biosystems, distributed by Life Technologies) as described previously (Umer et al., 2021 (link)). At the end of each PCR run, a melting point analysis was performed. Gapdh was used as reference gene for data normalization.