Development of a 3':5' digital PCR assay to determine horse mRNA integrity

Charis Du Cheyne; Yao Chen; Jurgen De Craene; Olivier Thas; Ward De Spiegelaere

doi:10.1016/j.ab.2021.114217

Development of a 3':5' digital PCR assay to determine horse mRNA integrity

Anal Biochem. 2021 Aug 1:626:114217. doi: 10.1016/j.ab.2021.114217. Epub 2021 May 1.

Authors

Charis Du Cheyne¹, Yao Chen², Jurgen De Craene¹, Olivier Thas³, Ward De Spiegelaere⁴

Affiliations

¹ Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820, Merelbeke, Belgium.
² Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820, Merelbeke, Belgium; Department of Data Analysis and Mathematical Modelling, Ghent University, B-9000, Ghent, Belgium; Ghent University Digital PCR Consortium, Ghent University, B-9000, Ghent, Belgium.
³ Department of Data Analysis and Mathematical Modelling, Ghent University, B-9000, Ghent, Belgium; Ghent University Digital PCR Consortium, Ghent University, B-9000, Ghent, Belgium; Data Science Institute, I-BioStat, Hasselt University, B-3500, Hasselt, Belgium; National Institute for Applied Statistics Research Australia (NIASRA), University of Wollongong, NSW, 2500, Wollongong, Australia.
⁴ Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820, Merelbeke, Belgium; Ghent University Digital PCR Consortium, Ghent University, B-9000, Ghent, Belgium. Electronic address: ward.despiegelaere@ugent.be.

PMID: 33939972
DOI: 10.1016/j.ab.2021.114217

Abstract

Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysis of the mRNA integrity, but also of its quantity.

Keywords: 3’:5′ assay; Digital PCR; RNA integrity; RNA quality; dPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Horses
RNA / analysis*
RNA / genetics
RNA Stability*
RNA, Messenger / analysis
RNA, Messenger / chemistry*
RNA, Messenger / genetics
Real-Time Polymerase Chain Reaction

Substances

RNA, Messenger
RNA