Primescript 2 1st strand cdna synthesis

Total RNA was extracted using an RNeasy Mini kit (Qiagen, 74106) according to the manufacturer's instructions, and was reversely transcribed using the PrimeScript II 1st strand cDNA Synthesis (Takara, 6210A). A random primer (hexadeoxyribonucleotide mixture; pd(N)6;Takara,3801) (50 μM) was used to guide the reverse transcription. Real-time RT-PCR analysis was performed using a Power SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies) and an Applied Biosystems 7500 Real-Time PCR System. Relative mRNA levels were calculated by normalizing to the levels of endogenous Gapdh mRNA (internal control) or encoded exogenous Gfp cDNA using Microsoft EXCEL^®. The relative transcript levels of samples were compared to the control, and the fold-changes are demonstrated. For each experiment, qPCR was performed in triplicate. Primer sequences are listed in Supplementary Table S1.

Total RNA from the aforementioned tissues and cells was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Then, 1 μg total RNA was reverse transcribed into cDNA by PrimeScript™ II 1st Strand cDNA Synthesis (TaKaRa, Dalian, Liaoning, China). Finally, qPCR was performed using SYBR Green as the detection system (TaKaRa) in a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The sequences of the qPCR primers used are summarized in Additional file 6: Table S2.

Total RNA in spermatocytes was extracted using the RNeasy Mini kit (Qiagen, 74 106) according to the manufacturer's instructions, and was reversely transcribed using the PrimeScript II 1st strand cDNA Synthesis (Takara, 6210A). A random primer (hexadeoxyribonucleotide mixture; pd(N)6;Takara,3801) (50 µM) was used to guide the reverse transcription. RT‐qPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies) and an Applied Biosystems 7500 Real‐Time PCR System. Relative mRNA levels were normalized against endogenous Gapdh (internal control). Relative transcription levels were compared with those of the control, and fold change was determined. RT‐qPCR experiments were performed in triplicate. Primer sequences are listed in Table S2, Supporting Information.