Revertaid first strand cdna synthesis kit

Total RNA samples were extracted with the RNAiso plus reagent (Takara, Japan). After digestion with gDNA digester (Yeasen, China), cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Yeasen). The reference gene RP18 was chosen as internal control (Pfaffl et al., 2004 (link); Huggett et al., 2005 (link)). The reaction mixture consisted of 5 µl TB Green Premix Ex Taq II (Tli RNaseH Plus), 1 µl of cDNA, and 0.25 µl of forward and reverse primers (see Supplementary Table S2; 10 µM) in a final reaction volume of 10 µl. The qPCR protocol included an initial denaturation step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 30 s. The relative expression of β-Actin was calculated by the $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\mathrm{T}}}$ method (Livak and Schmittgen, 2001 ; Shi et al., 2013 (link)). All experiments were repeated in triplicate.

The uterine tissue samples were immediately placed in liquid nitrogen and stored at −80 °C for RT-qPCR. Frozen tissues from −80 °C were immediately homogenized in Trizol reagent according to the manufacturer’s instructions (Thermo Fisher, MA, USA). The integrity and concentration of the RNA were measured using a NanoDrop Spectrophotometer 1000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). Target gene expression was evaluated by RT-qPCR on a 700 Fast Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The primers used are listed in Table 1. The PCR cycling parameters were as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. Relative mRNA expression levels were determined using the comparative Ct (ΔΔCt) method.

Total RNA was isolated from mouse brain using the RNAprep Pure Kit (Qiagen, Mannheim, Germany) following the manufacturer's instructions. To remove residual DNA contamination, 1 mg of total RNA was treated with 50 units of DNase I (Yeasen, China) at 37℃ for 30 minutes. The purified RNA was reverse‐transcribed into cDNA using a Revert Aid First Strand cDNA Synthesis Kit (Yeasen). qPCR was performed with a SYBR Green Real‐time PCR Master Mix (Yeasen) in a StepOne Plus Real‐Time PCR System (Applied Biosystems) using the following cycling conditions: 94℃ for 2 minutes, followed by 40 cycles of 94℃ for 5 s, 56℃ for 15 s and 72℃ for 20 s. Fluorescence data were acquired at the 72℃ step and during the melting curve programme. GAPDH and beta‐actin served as the reference genes. Triplicate PCRs were performed for each of three independently purified RNA samples. Quantitative PCR primers were designed to amplify fragments of approximately 100‐200 bp (Table 1) using Primer‐BLAST online software.

Total RNA samples were extracted using the RNAiso plus regent (Takara, Kyoto, Japan). Additional genomic DNA was digested by gDNA digester (Yeasen, Shanghai, China); 1.0 μg RNA was used for synthesizing cDNA by using the RevertAid First Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). The RP18 was chosen as the reference gene [35 (link),36 (link)]. The 10 μL reaction mixture consisted of 5 μL TB Green Premix Ex Tap Ⅱ (TliRNaseH Plus) (Takara, Kyoto, Japan), 3.5 μL of nuclease-free water, 1 μL of cDNA, and 0.25 μL of forward and reverse primers (Supplementary Table S1). The qPCR protocol included an initial denaturation step at 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 5 s, 60 ℃ for 30 s and 72 ℃ for 30 s. The relative expression of β-Actin was calculated by the 2^−ΔΔct method [37 (link)]. All experiments were repeated at least three times.