Quantifying Gene Expression in Male Infertility

To examine the expression of genes obtained from the sequencing results, we determined their mRNA levels in NOA and normal spermatogenesis samples. According to the instructions, total RNA from six testicular tissues was extracted using RNA-easyTM Isolation Reagent (R701-02, Vazyme, Nanjing, China), and the RNA samples were synthesized into cDNA using a Hifair® II 1st Strand cDNA Synthesis Kit (11121ES60, Yeasen, Shanghai, China). We performed qRT-PCR and analyzed gene expression as previously described [29 (link)]. The GAPDH gene was used as the endogenous reference. Detailed information regarding primer sequences was shown in Supplementary Table 4.