Quantifying Antiviral Gene Expression

Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel)
according to the manufacturer’s protocol. cDNA was synthesized from 1
μg total RNA using the QuantiTect RT kit (Qiagen) according to
manufacturer’s instructions. qPCR was carried out using the ViiA7 qPCR
system with TaqMan reagents using TaqMan
primers/probes (Life Technologies) for IFNB,
IFNL1, ISG15, IFIT1,
MX1, OAS1, TNFA,
IL6, ZC3HAV1, CSTF2,
HPRT, GAPDH and custom-designed,
isoform-specific probes for ZAP-S and ZAP-L (Supplementary Table 1).
TaqMan primers/probes for IFNL2 and
IFNL3 were previously designed and tested for
specificity^{6 (link), 41}. Target gene expression was normalized
to HPRT or GAPDH housekeeping genes.

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Schwerk J., Soveg F.W., Ryan A.P., Thomas K.R., Hatfield L.D., Ozarkar S., Forero A., Kell A.M., Roby J.A., So L., Hyde J.L., Gale M J.r., Daugherty M.D, & Savan R. (2019). RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions. Nature immunology, 20(12), 1610-1620.

Publication 2019

NucleoSpin RNA kit QuantiTect RT kit TaqManprimers/probes

Cdna Gapdh Gene expression Housekeeping genes Ifnb Isoform Oas1 Primers Tnfa

Corresponding Organization :

Other organizations : University of Washington, University of California, San Diego, Virginia Mason Medical Center, Alpine Immune Sciences (United States)

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Variable analysis

independent variables

Total RNA isolation using the NucleoSpin RNA kit
CDNA synthesis from 1 μg total RNA using the QuantiTect RT kit

dependent variables

Expression of IFNB, IFNL1, ISG15, IFIT1, MX1, OAS1, TNFA, IL6, ZC3HAV1, CSTF2, HPRT, GAPDH, ZAP-S, and ZAP-L

control variables

HPRT and GAPDH housekeeping genes used for target gene expression normalization

controls

Positive control: IFNL2 and IFNL3 TaqMan primers/probes previously designed and tested for specificity

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