RNA extraction, RT-PCR, and quantitative PCR

Total RNA was isolated from rat testes, Sertoli cells, germ cells, and kidney using TRIzol reagent (Life Technologies) and used for RT-PCR (n = 3) with the corresponding primer pairs (Table 3) as described (69). The identity of PCR products was confirmed by direct DNA sequencing at Genewiz (South Plainfield, NJ). Quantitative PCR (qPCR) (n = 3) was analyzed by the QuantStudio™ 12K Flex real-time PCR System (Thermo Fisher Scientific, Waltham, MA) with PowerUp SYBR Green master mix (Applied Biosystems, Foster City, CA) with glyceraldehyde 3-phosphate dehydrogenase as an internal control for normalization (69). Specificity of the fluorescence signal was verified by both melting curve analysis and gel electrophoresis, and the expression level of the target gene was determined using the 2−^ΔΔCT method.

Primer Pairs Used for qPCR or RT-PCR to Assess the Steady-State mRNA Level of Target Genes