TY  - JOUR
TI  - Detection of Cytoplasmic and Nuclear Circular RNA via RT-qPCR
AU  - Tan, Ke- En
AU  - Ng, Wei Lun 
AU  - Ea, Chee- Kwee
AU  - Lim, Yat Yuen
VL  - 13
IS  - 17
PY  - 2023
DA  - 2023/09/05
SP  - e4798
C1  - Bio-protocol 2023;13:e4798
DO  - 10.21769/BioProtoc.4798
UR  - https://doi.org/10.21769/BioProtoc.4798
AB  - Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs.Key features• This protocol builds upon the method developed by Gagnon et al. (2014) and extends its application to circRNA study.• Protocol for amplification of low levels of circRNA expression.• Analysis takes into consideration the ratio of cytoplasmic RNA concentration to nuclear RNA concentration.Graphical overview
KW  - Subcellular fractionation
KW  - RT-qPCR
KW  - Circular RNA
KW  - circRNA
KW  - Cytoplasmic circRNA
KW  - Nuclear circRNA
KW  - Low-abundance circRNA
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False