Fast reverse transcription kit

mRNA was extracted from 150 oocytes using the Dynabeads^TM mRNA DIRECT^TM Purification Kit (Invitrogen) according to the manufacturer’s instructions. A fast reverse transcription kit (TIANGEN, Beijing, China) was used for the synthesis of cDNA by reverse transcription. Each 20 μL quantitative RT-PCR (qRT-PCR) mixed system included 8 μL of deionized water, 10 μL of SuperReal PreMix Plus (TIANGEN), 1 μL of cDNA, and 0.5 μL each of forward and reverse primers (10 mM). Gene expression was quantified with the Mastercycler ep realplex system (Eppendorf, Hamburg, Germany) using the 2^–ΔΔCt method with GAPDH as the internal standard. The PCR conditions were as follows: 95°C for 3 min; 45 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. All primers used are listed in Supplementary Table 1.

The extraction of total RNA from cumulus cells was performed using the TRIpure Isolation Reagent (Roche, Germany) and quantified in a NanoDrop 2000 spectrophotometer (Thermo, USA). cDNA synthesis was performed using 1 μg of RNA and a fast reverse transcription kit according to the manufacturer’s instructions (Tiangen Biotech, China).
The expression levels of DSB repair genes in cumulus cells were detected using a SYBR Green quantitative PCR protocol with GAPDH as an internal control gene. RT-qPCR was carried out in triplicate in a 20 μl reaction volume using SuperReal PreMix Plus (TIANGEN, China), with 8 μl of sterile-deionized water, 1 μl of cDNA and 0.5 μl of each of the upstream and downstream PCR primers (10 μM), in a Mastercycler1 ep realplex machine (Eppendorf, Germany). For each sample, the threshold cycle (CT) was calculated, and the fold-increase in the experimental sample relative to the control was analyzed using the 2-ΔΔCt method. The primer sequences are shown in Table 1.