Quantifying RNA Expression Using RT-qPCR

Cellular RNA was isolated using a GenElute Total RNA MiniPrep kit (Sigma). Following isolation and treatment with DNase (Sigma), RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad) containing 1 μg of sample RNA per reaction. RT-qPCR was conducted using IQ SYBR green SuperMix (Bio-Rad) in a Bio-Rad CFX96 Touch real-time PCR detection system as well as an Applied Biosystems StepOne Plus real-time PCR machine. A modified threshold cycle (ΔC_T) method was used to calculate gene expression using human actin for normalization. Primer sequences for actin, ZIKV, IFN-β, IFN-λ, and ISGs have been previously published (43 (link)). Additional primer sequences can be found in Table S2 in the supplemental material.

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Bramley J.C., Drummond C.G., Lennemann N.J., Good C.A., Kim K.S, & Coyne C.B. (2017). A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier. mSphere, 2(3), e00206-17.

Publication 2017

GenElute Total RNA MiniPrep kit DNase iScript cDNA synthesis kit IQ SYBR green SuperMix CFX96 Touch real-time PCR detection system StepOne Plus real-time PCR machine

Actin Cdna Cellular Dnase Gene expression Human Isolation Primer Real time pcr Sybr green Synthesis Touch Zikv

Corresponding Organization :

Other organizations : University of Pittsburgh, Children's Hospital of Pittsburgh, Johns Hopkins University

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Variable analysis

independent variables

Treatment of RNA samples with DNase (Sigma)

dependent variables

Gene expression of actin, ZIKV, IFN-β, IFN-λ, and ISGs

control variables

Use of human actin for normalization of gene expression

controls

Positive control: None specified
Negative control: None specified

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