Hifair 1st strand cdna synthesis kit

Total RNA was extracted from 50 oocytes or 5 × 10⁵ cells using the TRIzol reagent (Invitrogen, United States), following the manufacturer’s instructions, and cDNA synthesis was completed using the Hifair 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology Co., Ltd., China). RT-PCR was performed with the SYBR Green Master Mix (Yeasen Biotechnology Co., Ltd., China) with the ABI 7500 Real-Time PCR system (Applied Biosystems, United States). Mouse Zscan4 (Forward: 5′- GAG​ATT​CAT​GGA​GAG​TCT​GAC​TGA​TGA​GTG-3′ and Reverse: 5′- GCT​GTT​GTT​TCA​AAA​GCT​TGA​TGA​CTT​C-3′), Zfp352 (Forward: 5′- ACC​ACC​TCA​AAG​AAC​ACC​AG-3′ and Reverse: 5′- ACA​AGG​GAC​AAG​CGT​AGA​AC-3′), were normalized against Gapdh (Forward: 5′-TCT​TCC​AGG​AGC​GAG​ACC​C-3′ and Reverse: 5′-CGG​AGA​TGA​TGA​CCC​TTT​T-3′), and the quantification of the fold change was determined by the comparative CT method.

Human cervical cancer cells (HeLa and SIHa) were purchased from the China Center for Type Culture Collection. Cells were maintained in DMEM (Servicebio, China) complete medium with 100 U/mL penicillin/streptomycin (YEASEN Biotech Co. Ltd., China) and 10% fetal bovine serum (FBS; YEASEN Biotech Co. Ltd.) at 37°C in a humidified atmosphere containing 5% CO₂. Total RNA was extracted from the synovial membrane using TRIzol reagent, and cDNA synthesis was performed with a Hifair 1st Strand cDNA Synthesis kit (YEASEN Biotech Co., Ltd.) according to the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using Hieff qPCR SYBR Green Master Mix (YEASEN Biotech Co. Ltd.), and the gene expression levels were detected by an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., USA). Relative mRNA expression was calculated with the 2^−ΔΔCT method compared to GAPDH expression. The primers are listed in Supplementary Table S1.

TRIeasy™ Total RNA Extraction Reagent (Yeasen, Shanghai, China) was used for total RNA extraction, and then the total RNA was reverse transcribed to cDNA with the Hifair^® 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China) according to the product instruction. Hieff UNICON®Power qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) was used to conduct RT-qPCR experiment on a Bio-Rad CFX96 System (Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of amplification at 95 °C for 10 s and 60 °C for 30 s. Relative mRNA expression levels of DPM1/2/3 were measured based on the 2^−ΔΔCt method with 18S used for normalization. The significance of expression analysis was completed using Student’s t-test. Table 2 showed the primers we used in this study.