Quantitative RT-PCR Analysis of Viral Genes

RNA was extracted using TRIzol reagent (Life Technologies), and reverse transcription was performed with 3 μg of total RNA using oligo(dT) and RevertAid Reverse Transcriptase (Thermo Scientific), according to the manufacturer’s instructions. Gene amplification was performed with SuperReal PreMix SYBR Green (TIANGEN) using an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies), and the expression levels of all the genes were normalized to those of β-actin. Primers for each exon region of ZAP, NS1, and β-actin have been reported^{26 (link)}.

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Hu C., Liu Y., Lin Y., Liang J.K., Zhong W.W., Li K., Huang W.T., Wang D.J., Yan G.M., Zhu W.B., Qiu J.G, & Gao X. (2018). Intravenous injections of the oncolytic virus M1 as a novel therapy for muscle-invasive bladder cancer. Cell Death & Disease, 9(3), 274.

Publication 2018

TRIzol reagent RevertAid Reverse Transcriptase SuperReal PreMix SYBR Green Applied Biosystems 7500 Fast Real-Time PCR System

Actin Exon Gene amplification Genes Oligo Primers Reverse transcriptase Reverse transcription Sybr green Trizol

Corresponding Organization :

Other organizations : Third Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University, Sixth Affiliated Hospital of Sun Yat-sen University

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Variable analysis

independent variables

RNA extraction method (TRIzol reagent)
Reverse transcription protocol (using oligo(dT) and RevertAid Reverse Transcriptase)
Gene amplification method (SuperReal PreMix SYBR Green)
PCR platform (Applied Biosystems 7500 Fast Real-Time PCR System)

dependent variables

Expression levels of ZAP, NS1, and β-actin genes

control variables

Amount of total RNA used for reverse transcription (3 μg)
Normalization of gene expression levels to β-actin

controls

Positive control: Expression of β-actin gene
Negative control: Not explicitly mentioned

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