Perfectstart green qpcr supermix

Total peripheral blood cell RNA was extracted with TRIzol (Invitrogen, 15 596 026), and the cDNA was synthesized using the PrimeScript RT Master Mix Kit (TaKaRa, RR036A). The qPCR was carried out using PerfectStart^TM Green qPCR SuperMix (TransGen Biotech, AQ601) on a Real‐time PCR Detection System (Roche, LightCycle480 II). RPL13A was served as internal control. Primers (5’‐3’) are listed in Table S5.

To validate the RNA-seq data, four DEGs from four different groups were selected for qRT-PCR assay with three biological replicates. All primer pairs were designed using software Oligo7 (DBA Oligo Inc., CO, USA) and shown in Table S1. The RNA samples used for qRT-PCR analyses were the same ones used in the RNA-Seq. RNA from each sample was reverse transcribed using the EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (Beijing Transgen Biotech Co. Ltd., Beijing, China). Quantitative real-time PCR (qRT-PCR) was performed using the PerfectStart^TM Green qPCR SuperMix (Beijing Transgen Biotech Co. Ltd., Beijing, China) with the CFX Connect^TM Real-Time System (Bio-Rad Laboratories, Inc., CA, USA). The PCR was initiated at 95 °C for 3 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. Actin7 served as internal reference gene to normalize the expression data.

Total mRNA was extracted immediately using NucleoZOL as described in “RNA isolation User manual”. In brief, 500 μL NucleoZOL was added per 5 × 10⁶ cells or 50 mg tissue for lysing or homogenizing, and then 200 μL RNase-free water was added to the lysate, shaking, incubating, and centrifuged for 15 min at 12,000 g at room temperature. The supernatant was mixed with isopropanol in equal volume and centrifuged for 10 min at 12,000 g to precipitate RNA. 75% Ethanol was used to wash RNA and RNase-free water was added to dissolve the RNA pellet. The concentration of RNA was measured by nanodrop and its integrity was detected by agarose gel electrophoresis. RNA was used to synthesize cDNA immediately using Thermo Scientific RevertAid Mix, and cDNA was kept in -80°C or mixed with 2 × PerfectStartTM Green qPCR SuperMix (TransGen Biotech) and primers according to the manufacturer’s instructions. PCR was performed for 40 cycles and ABI QuantStudio3 was used to collect SYBR Green I signal. Abs Quant/2nd Derivative Max was used to calculate the Cp value. The relative levels of individual mRNAs were calculated after normalization to the ACTB mRNA level. DNase I-treated total RNA was used as a negative control to rule out possible contamination of the RNA sample with genomic DNA. The primer sequences are listed in Table S1.

Total RNA was extracted from Control, H₂S, H₂S + AS, and AS treated roots according to the standard protocol of Trizol (Invitrogen, CA, United States). Total RNA was tailed addition reaction and first strand cDNA synthesis employed TransScript^® miRNA RT Enzyme Mix and 2 × TS miRNA Reaction Mix (TransGen Biotech, Beijing, China). The cDNA template for miRNA target gene qRT-PCR was synthesized using the TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (TransGen Biotech, Beijing, China). PerfectStart^TM Green qPCR Super Mix was used for qRT-PCR (TransGen Biotech, Beijing, China). Fifteen miRNAs and nine target genes were selected to conduct qRT-PCR and then verify the miRNA expression revealed by the RNA-seq. qRT-PCR was performed following the method used by Li et al. (2020) (link). U6 and 18S rRNA were used as an internal standard to normalize the expression of miRNA and target genes. The roots from the Control treatment were taken as a reference sample and the relative expression level of genes was set to 1. All primers, including miRNAs and their targets in the qRT-PCR experiments, are shown in Supplementary Tables 3, 4.

RNA integrity was evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, United States) and agarose gel electrophoresis. The RNA to be tested was reverse transcribed into cDNA using a TransScript All-in-One First-Strand cDNA Synthesis SuperMIX for qPCR (ransgen biotech, Beijing, China) kit according to the manufacturer’s instructions. Using β-actin (KJ476139.1) as an internal reference gene, Shanghai Ouyi Biomedical Technology Co., Ltd. designed primers for seven randomly selected genes and internal reference genes using the Primer-BLAST online program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), which were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd. The PerfectStart^TM Green qPCR SuperMix (transgen biotech, Beijing, China) kit was used to react on a LightCycler^® 480 II fluorescence quantitative PCR instrument (Roche, Swiss). The amount of gene expression was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). The primer information of genes is shown in Table 1.

After LPS stimulation (0.1 μg/mL) for 12 h, the total mRNA of HBZY-1 cells was extracted by using the RNeasy Kit (50) Plus Mini Qiagen Kit (74134, Qiagen) according to the instructions. The cDNA was made by the reverse transcription kit (Trans Script^® All-in-One First-Strand cDNA Synthesis Super Mix for qPCR, TransGen Biotech, Beijing, China) in a 20 μL reaction system. The amplification of each target gene was carried out in a 25 μL reaction system by using the Perfect Start^TM Taq DNA Polymerase kit (TransGen Biotech, Beijing, China) and observed through agarose gel electrophoresis. The real-time quantitative PCR was performed by using the LightCycler^® 96 Real-Time PCR System (Roche, Basel, Switzerland) and PerfectStart^TM Green qPCR SuperMix (TransGen Biotech, Beijing, China). The results were quantitatively analyzed by a 2^−ΔΔCt method. GAPDH was used as the internal control, and the primers sequences are shown in Table 1.