Hiscript 2 q rt supermix kit

Sub-samples (total RNA of 500 ng) of RNA-seq were treated with gDNA wiper Mix to remove genomic DNA, and then, cDNA was synthesized using Hiscript®II QRT SuperMix Kit (Vazyme, Nanjing, China) in accordance with the manufacturer’s protocols. The qRT-PCR was conducted using AceQ®qPCR SYBR®Green Master Mix (Vazyme, Nanjing, China) in accordance with the manufacturer’s instructions and run on an ABI 7300 Real Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The PCR program was as follows: 95 °C for 5 min and 40 cycles of 95 °C for 10 s, followed by 60 °C (± 5 °C) for 31 s. Primer 5.0 was used to design primers, the specificity of the primer sequences was tested using a Primer-BLAST of the NCBI database, and the reasonably designed primer sequences were sent to Sangong Bioengineering (Shanghai, China) Co., Ltd. for synthesis and purification (Additional file 2: Table S14). The ACTIN gene was used as the internal control. The application of the 2^{[–ΔΔC(t)]} method converts the threshold cycle value output by the instrument into the relative gene expression level [96 (link)]. Each reaction included two biological replications with three technical replications.

HCV RNAs were isolated from S29 cells and culture supernatants using TRIzol/chloroform extraction procedures (Life Technologies). For reverse transcription (RT), 500 ng of RNA was used by following the manufacture’s protocol of the HiScript II Q RT SuperMix kit (Vazyme, China), which included thermal reactions of 42°C for 2 min, 55°C for 15 min and 85°C for 5 s. One microliter of cDNA (1:10 dilution) was applied to quantitative PCR (qPCR) by using a StepOne qRT-PCR SYBR Green PCR Kit (Vazyme). HCV-specific qPCR was conducted in triplicate using a FastStart Universal SYBR Green Master (ROX) (Vazyme). The HCV-specific primers used in quantification was the following: HCV qS: 5′- CTTCACGCAGAAAGCGCCTA- 3′ and HCV qAS: 5′-CAAGCGCCCTATCAGGCAGT-3′ (Boson et al., 2011 (link)). Reactions were performed by one cycle of 95°C for 5 min, followed by 40 thermal cycles consisting of 95°C for 15 s and 60°C for 1 min. The amount of HCV RNA was calculated by a standard curve made from a serial dilution of a full-length HCV genomic plasmid, of which the copy number of DNA molecules was quantitated.

Total RNA was extracted from leaf tissues of maize, N. benthaniana, 16C, and Cucumis melo plants using the Transzol reagent (TransGen Biotech, Beijing, China) and then treated with a gDNA wipe enzyme (Vazyme, Nanjing, China) to eliminate the genomic DNA. The first-strand cDNA for RT-PCR was synthesized from 500 ng total RNA by HiScript^® II Q RT SuperMix kit (Vazyme, Nanjing, China) following the manufacturer’s instructions.
The qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a PCR machine (LC96, Roche, Basel, Switzerland). Gene-specific primers (Supplemental Table S1) were designed to amplify housekeeping genes of maize ZmUbi gene (GenBank accession: XM_008647047), N. benthamianaactin gene (GenBank accession: AY179605), and Cucumis meloEF1α gene (GenBank accession: XM_008459007). The accumulation levels of these genes are stable during viral infection and are therefore used as internal controls for qRT-PCR (Gao et al., 2012 (link); Sun et al., 2014 (link); Chen et al., 2017 (link)). Each qRT-PCR was performed with three biological replicates.

Total RNA was isolated from soybean and reverse transcribed by the HiScript^® II Q RT SuperMix Kit (Vazyme, China). Tissue expression was determined by qRT-PCR, using QuantStudio 5 (Applied Biosystems, United States), with AceQ^® qPCR SYBR^® Green Master Mix (Vazyme, China). A relative transcript level was calculated by the method of 2^–ΔΔCt (Livak and Schmittgen, 2001 (link)) and normalized to the GmTUBLIN (NM_001252709.2) transcript level. The primers are listed in Supplementary Table 1.

QRT-PCR was performed as previously described [22 (link), 23 (link)]. Briefly, total RNAs were isolated and purified with TRIzol reagent (Invitrogen, USA). Complementary DNA was synthesized from the total RNA using HiScript II Q RT Super Mix kit (Vazyme, Nanjing, China). QPCR was carried out with Step One Plus Real-Time PCR System (Applied Biosystems) using ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). PCR conditions were 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. PCR primers used were: p27, 5’-TCTCAGGCAAACTCTGAGGA-3’ (F) and 5’-CTTCCTCATCCCTGGACACT-3’ (R) [24 (link)]; β-actin, 5’-ACATCCGTAAAGACCTCTATGCCAACA-3’ (F) and 5’-GTGCTAGGAGCCAGGGCAGTAATCT-3’ (R). The mRNA expression level was normalized to the expression level of the housekeeping gene β-actin. All analyses were performed by using the 2^−ΔΔCt method [25 (link)].

We extracted STEC total RNA with Total RNA Extraction Reagent (Vazyme, R401) and got STEC cDNA with HiScript II Q RT SuperMix Kit (R222-01). Real-time quantitative PCR reactions were performed using hamQ Universal SYBR qPCR Master Mix Kit (Vazyme, Q711-00) and a StepOne real-time PCR system (Applied Biosystems, ABI StepOne). All measurements were duplicated and calculated using the arithmetic mean of Ct values. As an experimental control, the target gene Ct value was divided by the corresponding internal reference gene (GAPDH) Ct value. Using a relatively quantitative method, the obtained values were exponentiated 2^-ΔΔCt compared to the control 2^-ΔΔCt. The assay was performed in biological triplicate, and error bars represent SEM. Primers used for PCR are listed in the S2 Table.