SplintQuant: A method for accurately quantifying circular RNA transcript abundance without reverse transcription bias

Reverse transcription of RNA is fallible, introducing biases and confounding the quantification of transcript abundance. We demonstrate that circular RNAs (circRNAs) are more subjective to overestimation of transcript abundance than cognate linear RNAs due to their covalently closed, circular form,...

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Bibliographic Details
Published in:RNA (Cambridge) 2019-09, Vol.25 (9), p.1202-1210
Main Authors: Conn, Vanessa M, Conn, Simon J
Format: Article
Language:English
Subjects:
Abundance
Alternative splicing
Circular RNA
Deoxyribonucleic acid
DNA
Method
Oligonucleotides
Primers
Reverse transcription
Ribonucleic acid
RNA
RNA-directed DNA polymerase
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Summary:Reverse transcription of RNA is fallible, introducing biases and confounding the quantification of transcript abundance. We demonstrate that circular RNAs (circRNAs) are more subjective to overestimation of transcript abundance than cognate linear RNAs due to their covalently closed, circular form, producing multiple concatameric products from a single priming of reverse transcriptase. We developed SplintQuant, where custom DNA oligonucleotides are ligated by PBCV-1 DNA ligase only when bound to their target RNA. These circRNA-specific DNA oligonucleotides are terminally tagged with universal primers, allowing SplintQuant to accurately quantify even lowly-abundant circRNAs through highly-specific quantitative PCR (qPCR) in the absence of reverse transcription. SplintQuant is sensitive, specific, highly reproducible and applicable to the quantification of canonical and non-canonical RNA transcripts including alternative splice variants, gene fusions, and offers a gold-standard approach for accurately quantifying circRNAs.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.070953.119