Faststart universal sybr green master mix

The total RNA of the liver, adipose tissues (the WAT, the BAT, and the BeAT), and the small intestine (the duodenum, the jejunum, and the ileum) tissues were extracted using the TRIzol reagent (Takara, Japan) according to the instructions of the manufacturer. Reverse transcription was performed, and qRT-PCR was carried out on a 7,500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, United States), using the Roche FastStart Universal SYBR Green Master Mix (Roche Molecular Systems, Basel, Switzerland). β-Actin was chosen as the internal control and analyzed using the 2^−ΔΔCT method (primer sequences are provided in Supplementary Table S3).

Total RNA was extracted from rat myocardial tissues by using a high-purity total RNA rapid extraction kit (BioTeke Corporation, Beijing). According to the manufacturer's (BioTeke Corporation, Beijing) instructions, total RNA was reverse transcribed into cDNA for qPCR using the BioTeke Super RT Kit. Roche FastStart Universal SYBR Green Master Mix (Rox) was used for qRT-PCR through the first-step system. The mRNA level of each gene was normalized to the mRNA level of β-actin. The PCR conditions were as follows: initial activation at 50°C for 2 minutes and activation at 95°C for 10 minutes, followed by denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 minute for 40 cycles. The primers used for real-time PCR are listed in Table 1.

Total RNA from 60 MII oocytes was extracted by the RNeasy Mini Kit (Qiagen, Germany), combined with the RNase-free DNase (Qiagen) treatment to remove the genomic DNA. First strand cDNA was synthesized in a 20 μl reaction volume using the ABI kit (Life technologies). Primers were designed by the Primer-blast and shown in Supplementary Table S3. 10 µl PCR reaction volume, including 1 µl cDNA template, primers and Roche Fast Start Universal SYBR green master mix (Roche Molecular Systems), was used to quantify transcripts on a 7500 real-time PCR detection system (Applied Biosystems, Carlsbad, CA, USA). PCR parameters were set up as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. Each sample was tested in triplicate and Ywhag was used as the reference gene. Relative abundance of transcripts was calculated using the comparative Ct (2^−ΔΔCt) method.

Total RNA from C2C12 cells was isolated using Trizol reagent (Invitrogen). Following reverse transcription, qRT-PCR was conducted using a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and employing a Roche FastStart Universal SYBR Green Master Mix (Roche Molecular Systems; primer sequences are detailed in Table 1). Additionally, β-actin served as the internal control, and differential expression analysis was performed using the relative quantitative ΔCt method.

Denuded oocytes were washed twice with PBS (without Ca²⁺ and Mg²⁺) and stored at −80°C. Total RNA was extracted from 60 MII-stage oocytes from the control group, 60 MII oocytes from 3% DMSO treated (44h) group and 60 non-MII oocytes from 3% DMSO treated (44h) group (n = 3 replicates for each group), respectively, using RNeasy Mini Kit (Qiagen, USA) according to the manufacture's instructions. RNase-free DNase was added to remove genomic DNA. Total RNA from each sample was synthesized into cDNA in a 20μl final volume of reverse transcription system using the ABI kit following the instructions (Life Technologies). Primers were designed by primer-blast, as shown in S1 Table. Quantitative PCR was conducted in a 10μl reaction volume including 1μl cDNA template, primers and Roche FastStart Universal SYBR green master mix (Roche Molecular Systems) using a 7500 real-time PCR detection system (Applied Biosystems, Carlsbad, CA, USA). Thermal cycling parameters were setup as following: 95°C for 10min, followed by 40 cycles at 95°C for 15sec and 60°C for 1min. Transcripts were quantified in triplicates for each sample, and Ywhag was used as the reference gene. Relative abundance was calculated using the comparative Ct (2^−ΔΔCt) method [34 (link)].