A digital PCR-based protocol to detect and quantify RNA editing events at hotspots

Sunwoo Oh; Rémi Buisson

doi:10.1016/j.xpro.2022.101148

A digital PCR-based protocol to detect and quantify RNA editing events at hotspots

STAR Protoc. 2022 Mar 9;3(1):101148. doi: 10.1016/j.xpro.2022.101148. eCollection 2022 Mar 18.

Authors

Sunwoo Oh^{1

2}, Rémi Buisson^{1

2

3}

Affiliations

¹ Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92697, USA.
² Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA 92697, USA.
³ Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University of California Irvine, Irvine, CA 92697, USA.

Abstract

APOBEC3A, CRISPR programmable RNA base editors, or other enzymes can edit RNA transcripts at specific locations or hotspots. Precise quantification of these RNA-editing events is crucial to determine the activity and efficiency of these enzymes in cells. We have developed a quick method to quantify RNA-editing activity using digital PCR, a sensitive and quantitative technique to detect rare mutations by micro-partitioning bulk PCR reactions. This assay allows rapid absolute quantification of RNA editing events in cell lines or patient samples. For complete details on the use and execution of this protocol, please refer to Jalili et al. (2020) and Oh et al. (2021).

Keywords: CRISPR; Cancer; Cell Biology; Health Sciences; Molecular Biology.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems
Cytidine Deaminase
Gene Editing* / methods
Humans
Polymerase Chain Reaction
Proteins
RNA
RNA Editing* / genetics

Substances

Proteins
RNA
APOBEC3A protein, human
Cytidine Deaminase

Abstract

Publication types

MeSH terms

Substances

Grants and funding