How do you identify a circular RNA?
In single-end RNA-seq data, circRNAs can only be identified by reads aligned to the backsplice junction, as all other reads may have been generated by either a linear or a circular isoform.
What is circular nucleic acid?
Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner.
What is Backsplicing?
Circular RNAs (circRNAs) are generated by a specific type of splicing called back-splicing, wherein the 5′ terminus of a pre-mRNA upstream exon is non-colinearly spliced with the 3′ terminus of a downstream exon (Fig. 1).
What are divergent primers?
Divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.
What is RNase R treatment?
Depletion of rRNA coupled with depletion of poly(A)+ RNA or exonuclease (RNase R) treatment to deplete the majority of linear RNAs are the most favored methods to enrich for circRNAs among a pool of total RNA; these enriched circRNAs are then used for RNA-seq, microarray or RT-PCR analyses to detect and quantify true …
What contains circular RNA?
Location of circRNAs in the cell In the cell, circRNAs are predominantly found in the cytoplasm, where the number of circular RNA transcripts derived from a gene can be up to ten times greater than the number of associated linear RNAs generated from that locus.
What contains circular DNA?
Circular bacterial chromosomes. Mitochondrial DNA (mtDNA) Chloroplast DNA (cpDNA), and that of other plastids.
How are circular RNAs formed?
Circular RNAs can be produced by the direct ligation of 5′ and 3′ ends of linear RNAs, as intermediates in RNA processing reactions, or by “backsplicing,” wherein a downstream 5′ splice site (splice donor) is joined to an upstream 3′ splice site (splice acceptor).
What is the function of Exoribonuclease?
An exoribonuclease is an exonuclease ribonuclease, which are enzymes that degrade RNA by removing terminal nucleotides from either the 5′ end or the 3′ end of the RNA molecule.
How are circular RNA made?
Circular molecules are produced by introns produced from spliceosomal splicing, tRNA splicing, and group I and group II (self-splicing ribozymes) introns. Group I introns form circRNAs through autocatalytic ribozymal action, and while they can be detected in vivo, their function is yet to be determined.
What is the dephosphorylation step in RNA extraction?
This dephosphorylation step is unnecessary for RNAs that have not been cleaved in cis or in trans to generate a homogeneous 3′-end. If the RNA has been cleaved, the remaining cyclic 2′, 3′-phosphate product on the end of the RNA must be removed before any base additions can take place.
What are the challenges in the identification and quantification of circular RNA?
Detection and quantification of circular RNAs (circRNAs) face several significant challenges, including high false discovery rate, uneven rRNA depletion and RNase R treatment efficiency, and underestimation of back-spliced junction reads.
What is circular DNA amplification?
Circular DNA molecules for amplification are generated from circular RNA molecules. A method of producing a target RNA using a circular DNA, wherein said method does not comprise a step of linearizing said circular DNA.
Does rfxcas13d deplete circRNAs without depleting linear RNAs?
Only BSJ-gRNAs could guide RfxCas13d to deplete circRNAs without affecting linear RNAs, whereas other gRNAs that did not contain BSJ complementary sequences could direct RfxCas13d to degrade both circular and linear RNAs due to totally overlapping sequences.