Next Article in Journal
Antioxidant Versus Pro-Apoptotic Effects of Mushroom-Enriched Diets on Mitochondria in Liver Disease
Previous Article in Journal
Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?
Previous Article in Special Issue
LncRNAs Regulatory Networks in Cellular Senescence
Article Menu

Export Article

Open AccessArticle

Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants

1
Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India
2
School of Biotechnology, KIIT University, Bhubaneswar 751023, India
3
Laboratory of Genetics and Genomics, National Institute on Aging, NIH, Baltimore, MD 21224, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(16), 3988; https://doi.org/10.3390/ijms20163988
Received: 9 July 2019 / Revised: 29 July 2019 / Accepted: 31 July 2019 / Published: 16 August 2019
(This article belongs to the Collection Regulation by Non-Coding RNAs)
  |  
PDF [2223 KB, uploaded 16 August 2019]
  |     |  

Abstract

High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function. View Full-Text
Keywords: circRNAs; backsplice sequence; divergent primers; RNase R; RT-PCR; alternative splicing; splice variants circRNAs; backsplice sequence; divergent primers; RNase R; RT-PCR; alternative splicing; splice variants
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

Supplementary material

SciFeed

Share & Cite This Article

MDPI and ACS Style

Das, A.; Rout, P.K.; Gorospe, M.; Panda, A.C. Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants. Int. J. Mol. Sci. 2019, 20, 3988.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top