quantification-of-circular-rnas-using-digital-droplet-pcr

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Institute of Life Sciences

KIIT University

Skeletal muscles have an immense ability to regenerate from the muscle stem cells called satellite cells. The process of skeletal muscle regeneration is called myogenesis, which starts with activation of quiescent satellite cells immediately after muscle injury followed by proliferation and fusion of myoblasts into myotubes. Myogenesis is orchestrated through the expression of a specific set of genes which, at each step regulated by complex gene regulatory networks. Besides the well-established roles of transcription factors, increasing evidence demonstrated that circular (circ)RNAs modulate gene expression during myogenesis and are involved in muscle-related diseases. Here we review the recent findings of circRNAs involved in myogenesis.

Circular RNAs in myogenesis.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic β-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 () gene and are termed as circular intronic (). The expression of  transcripts in mouse pancreatic islets, and βTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of  was altered in βTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with  and their flanking region, suggesting their role in the  function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with . Gene ontology and pathway analysis of genes targeted by miRNAs associated with  suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially  transcripts, may regulate β-cell function and may play a critical role in the development of diabetes.

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Functional proteins in the cell are translated from the messenger RNA (mRNA) molecules, constituting less than 5% of the cellular transcriptome. The majority of the RNA molecules in the cell are noncoding RNAs, including rRNA, tRNA, snRNA, piRNA, lncRNA, microRNA, and poorly characterized circular RNAs (circRNAs). Recent studies established that circRNAs regulate gene expression by associating with RNA-binding proteins and microRNAs. With the growing understanding of circRNA functions, a subset of circRNAs has been reported to translate into proteins. Interestingly, the presence of Open Reading Frames (ORFs), N6-methyladenosine (m6A) modifications, and internal ribosomal entry sites (IRES) in the circRNA sequences indicate their coding potential through the cap-independent translation initiation mechanism. The purpose of this review is to highlight the mechanism of circRNA translation and the importance of circRNA-encoded proteins (circ-proteins) in cellular physiology and pathology. Here, we discuss the computational and molecular methods currently utilized to systematically identify translatable circRNAs and the functional characterization of the circ-proteins. We foresee that the ongoing and future studies on circRNA translation will uncover the hidden proteome and their therapeutic implications in human health. This article is categorized under: RNA Methods > RNA Analyses in Cells Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Translation > Mechanisms.

Circular RNA translation, a path to hidden proteome.

Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect on downstream target genes and cellular physiology. Biochemical assays, including reporter assays, AGO2 pulldown, ribonucleoprotein pulldown, and biotin-labeled RNA pulldown, are used to capture the association of miRNAs and RBPs with circRNAs. Only a few studies have used circRNA pulldown assays to capture the associated miRNAs and RBPs under physiological conditions. In this detailed protocol, the circRNA of interest () was captured using a biotin-labeled antisense oligo (ASO) targeting the  backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads. The specific enrichment of circRNA was analyzed using reverse transcription quantitative PCR (RT-qPCR). Furthermore, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA. In summary, the specific pulldown of circRNA using this quick and easy method makes it a useful tool for identifying and validating circRNA interaction with specific miRNAs and RBPs.

Antisense Oligo Pulldown of Circular RNA for Downstream Analysis.

High-throughput RNA-sequencing (RNA-seq) technologies combined with novel bioinformatic algorithms discovered a large class of covalently closed single-stranded RNA molecules called circular RNAs (circRNAs ). Although RNA-seq has identified more than a million circRNAs, only a handful of them is validated with other techniques, including northern blotting, gel-trap electrophoresis, exonuclease treatment assays, and polymerase chain reaction (PCR). Reverse transcription (RT) of total RNA followed by PCR amplification is the most widely used technique for validating circRNAs identified in RNA-seq. RT-PCR is a highly reproducible, sensitive, and quantitative method for the detection and quantitation of circRNAs. This chapter details the basic guidelines for designing suitable primers for PCR amplification and validation of circRNAs .

Debojyoti Das

Institute of Life Sciences

Quantification of Circular RNAs Using Digital Droplet PCR