RT-PCR testing

mRNA expressed from the SPAG9 gene in tissues was quantified as previously described (12). Total RNA was isolated and cDNA was synthesized using a RevertAid M-MulV First Strand cDNA Synthesis kit (Thermo Fisher) in accordance with the supplier's protocol. RT-PCR was performed using SPAG9-specific primers. Primers were designed using software Primer 3.0 and were synthesized by China Yuantai Company. Primers were: homo-SPAG9 forward, 5′-AGCCGACTTTTCAGCTCCTC-3′ and reverse, 5′-AAAGCCTGCACTCTACCGTC-3′. Expected fragment length was 114 bp, and the predicted melting temperature was 59°C. The GAPDH mRNA was amplified as an internal control with primers homo-GAPDH forward, 5′-caatgaccccttcatt-gacc-3′ and reverse, 5′-gacaagcttcccgttctcag-3′. The expected fragment length was 106 bp. The real-time PCR results were analyzed with SDS 7900 software (ABI).