دورية أكاديمية
Detection of Cytoplasmic and Nuclear Circular RNA via RT-qPCR.
| العنوان: | Detection of Cytoplasmic and Nuclear Circular RNA via RT-qPCR. |
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| المؤلفون: | Tan KE; Institute of Biological Sciences, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia., Ng WL; Institute of Biological Sciences, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia., Ea CK; Institute of Biological Sciences, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia., Lim YY; Institute of Biological Sciences, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia. |
| المصدر: | Bio-protocol [Bio Protoc] 2023 Sep 05; Vol. 13 (17), pp. e4798. Date of Electronic Publication: 2023 Sep 05 (Print Publication: 2023). |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Bio-protocol LLC Country of Publication: United States NLM ID: 101635102 Publication Model: eCollection Cited Medium: Internet ISSN: 2331-8325 (Electronic) Linking ISSN: 23318325 NLM ISO Abbreviation: Bio Protoc Subsets: PubMed not MEDLINE |
| أسماء مطبوعة: | Original Publication: Sunnyvale, CA, USA : Bio-protocol LLC, [2011]- |
| مستخلص: | 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. Competing Interests: Competing interestsThe authors declare no competing interests. (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.) |
| References: | Nat Biotechnol. 2014 May;32(5):453-61. (PMID: 24811520) Nat Protoc. 2014 Sep;9(9):2045-60. (PMID: 25079428) Nat Rev Genet. 2016 Oct 14;17(11):679-692. (PMID: 27739534) Brief Bioinform. 2021 Mar 22;22(2):2182-2190. (PMID: 32349124) |
| فهرسة مساهمة: | Keywords: Circular RNA; Cytoplasmic circRNA; Low-abundance circRNA; Nuclear circRNA; RT-qPCR; Subcellular fractionation; circRNA |
| تواريخ الأحداث: | Date Created: 20231018 Latest Revision: 20231020 |
| رمز التحديث: | 20231021 |
| مُعرف محوري في PubMed: | PMC10577455 |
| DOI: | 10.21769/BioProtoc.4798 |
| PMID: | 37849784 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 2331-8325 |
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| DOI: | 10.21769/BioProtoc.4798 |