Hiscript 3 rt supermix with gdna wiper

According to the manufacturer’s instructions, TransZOL (TransGEN Biotech, Beijing, China) was used to extract the Total RNA from the leaves of wheat seedlings. Used DeNovix DS-11 spectrophotometrically to check the quality and quantity of Isolated RNA’s and perform 2% agarose gel electrophoresis, and stored at −80°C for further use. According to the manufacturer’s instructions, we used HiScript^®III RT SuperMix with + gDNA wiper (Vazyme, Nanjing, China) for cDNA synthesis. Quantitative real-time PCR (qRT-PCR) was performed using CFX96 Real-Time PCR Detection System (Bio-Rad, United States) and AceQ^® Universal SYBR^® qPCR Master Mix (Vazyme, Nanjing, China). The total reaction mixture for qRT-PCR was 20 μl including 10 μl 2 × AceQ Universal SYBR qPCR Master Mix, 2 μl cDNA, 1 μl forward primer (10 μmol), 1 μl reverse primer (10 μmol), and 6 μl RNAase free water. The parameters followed by qRT-PCR are 95°C for 5 min, and then 40 cycles of 95°C for 10 s and 60°C for 30 s. The wheat β-actin gene was used as an internal control for all qRT-PCR expression analysis (Hu et al., 2013 (link)). The 2^–ΔΔCT method was applied to determine relative gene expression level (Livak and Schmittgen, 2001 (link)). In all our experiments we used three biological repeats and three technical repeats for all qRT-PCR analysis. All the primers used in qRT-PCR are listed in Supplementary Table 1.

Total RNA was isolated with UNlQ-10 Column TRIzol Total RNA Isolation Kit (Sangon Biotechnology, Shanghai, China) and reversely transcribed into cDNA using HiScript III RT SuperMix with gDNA wiper (Vazyme, Nanjing, China). Gene expression levels were quantified by an ABI 7,500 Sequence Detector (Applied Biosystem, Carlsbad, CA, USA) with the AceQ qPCR SYBR Green Master Mix (Vazyme Biotechnology, Nanjing, China). Primer sequences synthesized by GENEWIZ were shown in Table 1. Reactions were carried out with 2.8 μl reverse transcribed RNA and gene-specific primers in a total volume of 20 μl. PCR conditions consisted of an initial incubation step of 2 min at 50°C and a heat activation step of 10 min at 95°C. This was followed by 40 cycles of denaturation at 95°C for 10 s and annealing and extension at 60°C for 30 s.

Total RNAs from pig spleen tissues were extracted using Trizol (TaKaRa, Dalian, China). Then, reverse transcription of RNA was conducted using HiScript III RT SuperMix with gDNA wiper (Vazyme, Nanjing, China). The RT-qPCR reaction system contained 5 μl SYBR Green Mixture (Vazyme, Nanjing, China), 1 μl of the cDNA template, 0.2 μl of each primer, and 3.6 μl deionised water. The thermal conditions were as follows: 95°C for 5 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s. The GAPDH genes were set as the internal controls. All the forward and reverse primers for the RT-qPCR assays are listed in Table 1. The expression level of each validated gene for each timepoint was calculated by the 2^−ΔΔCt method.

RNA-seq data of mice (GSE98150) were downloaded from Gene Expression Omnibus. Gene expression of MAPKs during early embryo development was normalized with log2(count + 1).
Blastocysts (30 for each treatment) were collected at day E4, and mRNA was purified using Dynabeads mRNA DIRECT™ KIT (invitrogen) following the manufacturer’s instructions. Random primed reverse transcription was performed using HiScript III RT SuperMix with gDNA wiper (R323-01, Vazyme, Nanjing, China). cDNA was added to 10 µl of quantitative PCR mix (Q111-02, Vazyme, Nanjing, China). RT-PCR reactions were performed on a Step-One Plus Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). The primers for quantitative analysis are shown in Additional file 2: Table S1. Gene expression was calculated using the 2^ΔΔct method, and GAPDH was used for normalization as endogenous reference genes.

Total RNA was extracted from the collected tissues using TRIzol reagent (Invitrogen, USA). First-strand cDNA was synthesized using a HiScript III RT SuperMix with gDNA wiper (Vazyme Biotech Co., Ltd) according to the manufacturer’s protocol. The expression levels of chicken ELOVLs and other genes were detected using quantitative real-time PCR (qRT-PCR). The specific primers were designed by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primerblast/) and synthesized by Sangon Biotech Co. Ltd. (Shanghai, China) (Additional file 9: Table S7). All reactions were carried out in triplicate. The relative level of each mRNA was normalized to the housekeeping gene β-actin and analysed by using the 1000 × 2^−Δct method.