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Abstract

Genetics

R-Loop Analysis by Dot-Blot

Published: January 22nd, 2021

DOI:

10.3791/62069

1Life Sciences Institute, University of Michigan, Ann Arbor, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 3Department of Pediatrics, University of Michigan, Ann Arbor, 4Howard Hughes Medical Institute, 5Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health

Abstract

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.

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