Dataset Information

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

ABSTRACT: High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.

SUBMITTER: Panda AC

PROVIDER: S-EPMC5499592 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

Panda Amaresh C AC De Supriyo S Grammatikakis Ioannis I Munk Rachel R Yang Xiaoling X Piao Yulan Y Dudekula Dawood B DB Abdelmohsen Kotb K Gorospe Myriam M

Nucleic acids research 20170701 12

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enri ...[more]

PMID: 28444238

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Dataset Information

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

Publications

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

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