Hifair 3 1st strand cdna synthesis supermix for qpcr gdna digester plus

Samples of algal cells grown under the photoheterotrophic and the autotrophic conditions were harvested and were used for qPCR validation. RNA extraction and RNA quality check were conducted as previously described. The Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus, Yeasen, Shanghai, China) was used for reverse transcription of 1 µg of total RNA according to manufacturers’ instructions. The quantitative real-time PCR (qRT-PCR) for gene expression assay was performed using Hieff® qPCR SYBR® Green Master Mix (No Rox) (Yeasen, Shanghai, China) on a quantitative thermal cycler (Light Cycler 480II, Roche, Basel. Switzerland). Specific primer pairs were designed for each target gene through Primer Premier (version 5.0) software (PREMIER Biosoft, San Francisco, CA, USA) using known sequences in the NCBI database (Supplemental Table S3). The qRT-PCR conditions were as follows: preincubation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, annealing temperature (corresponding specific primer pairs) for 20 s, and 72 °C for 20 s. Melting curves were systematically monitored (temperature gradient at 0.5 °C/s from 60 to 95 °C). Using 18S as reference genes, the genes were quantified using the 2^−∆∆Ct value method according to Pfaffl (2001 (link)). The value of algal cell in the autotrophic condition was assigned as an arbitrary value of 1.

Total RNAs of cells were extracted using TRNzol Universal (Tiangen Biotech) according to the manufacturer's guidelines. Then cDNA synthesis was performed using Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA Digester Plus) (Yeasen Biotechnology). Then qRT‐PCR was performed with QuantStudio5 (Thermo Fisher Scientific) using Hieff^® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen Biotechnology) based on the manufacturer's guidelines. We used GAPDH as the endogenous control and the 2^–ΔΔCT method to calculate the gene expression.

The total RNA of mouse mammary gland tissue and MAC-T cells was extracted with TRIzol reagent (Solarbio, Beijing). Q5000 ultramicro spectrophotometer (Quawell, USA) was used to evaluate RNA purity and measure RNA concentration. Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) and Hieff® qPCR SYBR® Green Master Mix were acquired from Yeasen (Shanghai, China) and used to examine target gene expression by qPCR with 2^−ΔΔCq method. The primers used in this study are shown in Table 1.

To verify the PCO gene expression in different tissues, samples of root, stem, leaf, flower, silique and seed from Arabidopsis and B. napus, and samples of root, stem and leaf from B. rapa and B. oleracea were collected. Leaf and root samples of B. napus under waterlogging stress treatment with 6 h and 12 h were collected as well. Total RNA was extracted using MolPure^® Plant RNA Kit according to manufacturer instructions (Yeasen, Shanghai, China). The first strand cDNA was synthesized by Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen, Shanghai, China). Then, the gene relative expression was detected by qRT-PCR assay using PerfectStart^® Green qPCR SuperMix (TransGen, Beijing, China) and a CFX96™ Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). Gene expression was normalized to AtActin2, BraGAPDH, BoActin and BnaActin7, respectively [27 (link),53 (link),54 (link),55 (link)]. Relative gene expression values were calculated with the ΔΔCt method. Experiments were performed with three biological replicates. Primers for qRT-PCR were listed in Table S6.

To detect the expression of BATF38, IRF5, ZBTB38 in A549 and H358 cell lines, RT-qPCR was carried out on an QuantStudio^® 5 real-time PCR system (Applied Biosystems) with proper PCR parameters.
Total RNAs were extracted by TRIzol (TIANGEN, Beijing, China). The first-strand cDNA was synthesized using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, Tokyo, Japan) according to the manufacturer's instructions. Then Hieff^® qPCR SYBR Green Master Mix (Low Rox Plus) (YEASEN) was used with the following PCR parameters, 1 cycle of 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 34 s at 60 °C. β-actin was used as the reference. Primers used in this study are listed in Additional file 7: Table S2.
All the samples were repeated three times.

The total RNA of longan EC, ICpEC, GE, and EC treated with different hormones were extracted by TransZolUp kit (TransGen Biotech), and the total RNA of different development stages of the zygotic embryo was extracted by BioTeke kit (Cat. No. RP3301). The cDNA was synthesized with a Hifair^®III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen). Primer design was performed using Primer3 online software (https://primer3.ut.ee/cgi-bin/primer3/primer3web_results.cgi) (Supplementary Table 1). DlACTB, DlEF-la, and DlUBQ were used as internal control genes (Lin and Lai, 2010 (link)). The qPCR system was 20 μL, including HRbio™ qPCR SYBR^®Green Master Mix (No Rox) (Heruibio), 8.2 μL ddH₂O, 1 µL of 10-fold diluted cDNA, and 0.4 μL specific primer pairs, with three replicates. The reaction procedure was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 58°C for 30 s, and 2^−ΔCT was used to calculate genes expression (Livak and Schmittgen, 2002 (link); Vandesompele et al., 2002 (link)). SPSS software was used for significant analysis of gene differences, and GraphPad 8.0.2 was used for the draft.