Mirna specific primer

Total RNA was isolated from sperm or GC2 cells using a Total RNA Isolation Kit (BEI‐BEI Biotech), and from embryos using an RNA Isolation Kit (cat. no., KIT0204; Invitrogen; Thermo Fisher Scientific, Inc.). For miRNA detection, complementary DNA (cDNA) was synthetized from total RNA using miRNA‐specific primers (RiboBio). For mRNA detection, cDNA was synthetized from total RNA using PrimeScript^® RT Master Mix (Takara) reverse transcriptase, according to the manufacturer's instructions. cDNA was quantitated using RT‐qPCR with a Roche LightCycler^® 96 Real‐time PCR system (Roche Diagnostics). Real‐time PCRs were performed in triplicate. β‐actin and U6 were used as endogenous controls for mRNA and miRNA, respectively. Relative expression was calculated using the comparative ∆∆Ct method. The primers for miRNA real‐time PCR were purchased from RiboBio Company. The other primer sequences are presented in Table S1.

Total RNA was isolated from cell lines with TRIzol (Invitrogen Life Technologies, Carlsbad, USA) according to the manufacturer’s protocol. A NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was used to measure RNA concentration. Reverse transcription reactions were performed with PrimeScript RT Reagent kit (Takara, Dalian, China), and quantitative real-time PCR (qRT-PCR) was performed using FastStart Universal SYBR Green Master (Roche, Mannheim, Germany) and the Step One Plus Real-time PCR system (Applied Biosystems, Singapore, Singapore). Expression was normalized to β-actin level. Primer sequences were as followed in Supplemental Table I. For miR-145 detection, miRNA isolated from cell lines using miRNA extract kit (Haigene, Haerbing, China), according to the manufacturer’s protocol. Reverse transcription reactions were performed with RevertAid First Strand cDNA Synthesis Kit (Thermo scientific, Lithuania, EU). miRNA-specific primers were purchased from RiboBio (RiboBio, Guangzhou, China), and the relative miR-145 expression level was normalized to U6. The 2-ΔΔCt method was used for relative quantification. All experiments were performed in biological triplicates, each in triplicates (n = 3).

Total RNAs were extracted by TRIzol Reagent (Invitrogen). Total RNAs (2mg) was reverse transcribed using a reverse transcription kit (Thermo scientific). The miRNA specific primers (Ribobio) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo scientific) were used for qRT-PCR to examine the relative quantification of miRNAs with the 7900HT Fast Real-Time PCR system (Applied Biosystems) and U6 was used as endogenous control. Each reaction was performed in triplicate, and analysis was performed by the 2^−ΔΔCt method.

The total RNA was isolated using RNAiso (Takara, Dalian, China). miRNA was subsequently reverse‐transcribed to cDNA using the miRNA‐specific stem‐loop reverse‐transcription primer (Ribobio, Guangzhou, China). The amount of target gene expression (2^−ΔΔCt) was normalized via the endogenous small nuclear RNA U6 using miRNA‐specific primers (Ribobio). The reaction conditions were performed according to the instructions from Ribobio Co., Ltd with SYBR Green qPCR Mix (BioRad, Hercules, CA).

The total RNA was isolated using RNAiso (Takara, China). miRNA was subsequently reverse-transcribed to cDNA using the miRNA-specific stem-loop reverse-transcription primer (Ribobio, China). The relative expression levels of miRNA were calculated using the 2^-ΔΔCt method (25 (link)) and normalized to the internal control U6, using miRNA-specific primers (RiboBio, China). The reaction conditions were performed according to the instructions from Ribobio Co., Ltd with SYBR Green qPCR Mix (BioRad, USA).