RNA isolation, in vitro transcription, and biotin-label assay

Junji Xing; Ao Zhang; Yong Du; Mingli Fang; Laurie J. Minze; Yong-Jun Liu; Xian Chang Li; Zhiqiang Zhang

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RNA isolation, in vitro transcription, and biotin-label assay

JX Junji Xing

AZ Ao Zhang

YD Yong Du

MF Mingli Fang

LM Laurie J. Minze

YL Yong-Jun Liu

XL Xian Chang Li

ZZ Zhiqiang Zhang

This method is extracted from research article: Nat Commun, May 2021

Identification of poly(ADP-ribose) polymerase 9 (PARP9) as a noncanonical sensor for RNA virus in dendritic cells

DOI: 10.1038/s41467-021-23003-4

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Viral genomic dsRNA from reovirus was isolated with QIAamp Viral RNA Mini Kit (Cat No: 52904, Qiagen) and were biotin-labeled with Pierce RNA 3’ End Biotinylation Kit (Cat No: 20160, ThermoFisher Scientific). The isolated reovirus genomic RNA was used to synthesize cDNA with the iScript cDNA Synthesis Kit (Bio-Rad). The viral cDNA was used as template to synthesize viral PCR fragments (8 to 1187 bp of reovirus S4 gene; 11 to 1198 bp of reovirus S3 gene; 16 to 1320 bp of reovirus S2 gene; 9 to 1410 bp of reovirus S1 gene) by PCR. The reovirus dsRNA including Reo 1187, Reo1198, Reo1320, and Reo1410 were synthesized using above PCR fragments with HiScribe T7 High Yield RNA Synthesis Kit (Cat No: E2040S, New England BioLabs) without 5’ppp or capped and were biotin-labeled with Pierce RNA 3’ End Biotinylation Kit.

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