The mRNA expressions of Olfr2, iNOS, Adcy3, NLRP3, GSDMD, Caspase-1, Arg-1, NEK7, ASC, IL-1β, IL-18, and TNF-α were measured by RT-qPCR. Total RNA was isolated from Ana-1 cells, BMDMs, or aortas by Trizol reagent (R401–01, Vazyme, Nanjing, China). The isolated total RNA was reverse transcribed into complimentary (c)DNA via the HiScript II Q RT SuperMix (R223–01, Vazyme). The RT-qPCR was performed on StepOne™ Plus device (Applied Biosystems, Foster City, CA, USA) following the protocol of HiScript II One Step RT-qPCR SYBR Green Kit (Q221–01, Vazyme). The 2-ΔΔCT method was used to analyze the relative mRNA expression normalized to the reference gene GAPDH. All primers were synthesized by TSINGKE (Wuhan, China). The primer sequences of genes were presented in Table 1
Hiscript 2 one step rt qpcr sybr green kit
Manufactured by Vazyme
Sourced in China
The HiScript II One Step RT-qPCR SYBR Green Kit is a reagent kit for performing reverse transcription and quantitative real-time PCR in a single reaction. The kit utilizes SYBR Green I as the detection dye.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qiaamp viral rna kit, by Qiagen (2 mentions)
Hiscript 2 q rt supermix, by Vazyme (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Steponeplus device, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
6 protocols using hiscript 2 one step rt qpcr sybr green kit
1
Molecular Profiling of Inflammation Pathways
2
Quantification of HBV and Liver Transcripts
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HBV pgRNA, HBV encapsidated pgRNA, HNF1α, HNF3α, and HNF4α mRNAs in HepG2.2.15 cells were analyzed by RT-qPCR normalized to the level of the internal control gene, β-actin. The PCR analysis was run with HiScript II One-Step RT-qPCR SYBR Green Kit (Vazyme Biotech, Nanjing, Jiangsu, China) and Applied Biosystems 7500 Fast Dx Real-Time qPCR system according to the following thermocycling parameters below: 50 °C for 3 min, followed by 95 °C for 30 s and 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Sequences of the primers used are available upon request.
3
SARS-CoV-2 Viral Load Quantification
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Vero E6 cells were seeded in 12-well plates at a density of 2 × 105 cells/well. After SARS-CoV-2 infection, cells were incubated for an indicated time. Viral RNA in supernatant was isolated with the QIAamp Viral RNA Kit (Qiagen, 52906) following the manufacturer’s instructions. Virus copies were then detected by RT-qPCR methods with the HiScript II One Step RT-qPCR SYBR Green Kit (Vazyme Biotech, Nanjing, China) using an ABI 7500 real time PCR system (Applied Biosystems, CA, United States). The protocol for RT-qPCR was as follows: 50°C for 15 min, 95°C for 30 s, followed by 45 cycles at 95°C for 10 s and 63°C for 35 s. The specific primers used to detect the SARS-CoV-2 N gene were as follows: Forward: 5′-GGGGAACTTCTCCTGCTAGAAT-3′; Reverse: 5′-CAGACATTTTGCTCTCAAGCT-3′. The sequence of Taqman probe was as follows: 5′-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3′.
4
ACE2 Expression in Bacteria-Treated Vero Cells
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The expression level of ACE2 in Vero cells cocultured with bacteria was determined by RT-qPCR using a HiScript® II One Step RT-qPCR SYBR® Green kit (Vazyme, Nanjing, China) following the manufacturer’s instructions, with the primers ACE2-F (5′-CGAAGCCGAAGACCTGTTCTA-3′) and ACE2-R (5′-GGGCAAGTGTGGACTGTTCC-3′). Briefly, the amplification was performed as follows: 50 °C for 15 min, 98 °C for 2 min, followed by 40 cycles of 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 30 s), and a default melt curve step in ABI Stepone machine. The housekeeping gene GAPDH was used as the reference gene with the primers GAPDH-F (5′-AACTCTGGTAAAGTGGAT-3′) and GAPDH-R (5′-TACTCAGCGCCAGCATCG-3′). The relative expression level of ACE2 was determined using the 2−ΔΔCt method as described previously (Livak and Schmittgen 2001 (link)) and compared to that of the control group using the Student’s t test by R program (version 3.5.3; https://cran.r-project.org/bin/windows/base/old/3.5.3/ ).
5
SARS-CoV-2 Variant Infection and Quantification
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Vero E6 cells were seeded into 6-well plates at a density of 2 × 105 cells/well for 24 h. Then, SARS-CoV-2, Beta, and Delta variants were infected at a multiplicity of infection (MOI) of 0.008. The QIAamp Viral RNA Kit (Qiagen, 52,906) was used to extract the viral RNA in the supernatant. Viral copy numbers were detected by absolute quantitative RT-qPCR methodology using the HiScript II One Step RT-qPCR SYBR Green Kit (Vazyme Biotech, Nanjing, China) on an ABI 7500 real-time PCR system (Applied Biosystems, CA, USA). The protocol for RT-qPCR was as follows: 50 °C for 15 min, 95 °C for 30 s, followed by 45 cycles at 95 °C for 10 s and 63 °C for 35 s. The specific primers used to detect the N and E genes were as follows:
N gene Forward: 5′-GGGGAACTTCTCCTGCTAGAAT-3′;
Reverse: 5′-CAGACATTTTGCTCTCAAGCT-3′;
E gene Forward: 5′-CGATCTCTTGTAGATCTGTTCTC-3′.
Reverse: 5′-ATATTGCATTGCAGCAGTACGCACA-3′.
N gene 5′-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3′.
E gene 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3′.
6
Quantitative Gene Expression Analysis
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The RNA extraction was performed using RNAiso plus (Takara, Dalian, Liaoning, China) according to manufacturer's protocols, then utilize the PrimeScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, Jiangsu, China) to synthesize cDNA based on the manufacturer’s manual. The RT-qPCR analysis was conducted using HiScript II One Step RT-qPCR SYBR Green Kit (Vazyme, Nanjing, Jiangsu, China) based on the manufacturer’s manual by the StepOne Plus Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). The primers used in this study were designed using the NCBI Primer-Blast tool. All primer sequences for target genes are listed in Supplementary Table S1 . The fold changes were calculated after normalizing to the housekeeping gene β-actin, and the 2−ΔΔCt method was used to estimate mRNA abundance [36 (link)]. All experiments were performed in triplicate.
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