Identification of Circular RNAs by Multiple Displacement Amplification and Their Involvement in Plant Development

Guria, Ashirbad; Sharma, Priyanka; Natesan, Sankar; Pandi, Gopal

doi:10.1007/978-1-0716-1645-1_4

Cited by 2 publications

(1 citation statement)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Apart from microarrays (Zhang Z. et al, 2018;Li et al, 2019;Li et al, 2021;Zhong et al, 2021), circRNA ampli-seq panels, fluorescent padlock probes (Zaghlool et al, 2018) or the RNase H cleavage-based assay (Barrett and Salzman, 2016), and a few other methods require prior circRNA sequence information; dependency on NGS-based approaches is found to be the only viable option for de novo circRNA identification till date (Gao et al, 2015). This, which we termed as traditional RNA-Seq here, could be performed either by sequencing of total RNA or by poly(A)-depleted RNA or/and linear RNA-depleted RNA (Xiao and Wilusz, 2019) or rRNA-depleted RNA (Memczak et al, 2012;Salzman et al, 2012;Westholm et al, 2014;Gao et al, 2015) or combining two or all the approaches together (Pandey et al, 2019;Shao et al, 2019;Han et al, 2020;Guria et al, 2021). However, these strategies require a huge amount of NGS reads for detection of a handful of circRNAs (Jeck and Sharpless, 2014) as previously evidenced in the literature (Salzman et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method

Guria

Sharma

Srikakulam

et al. 2022

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5′–3′ backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from Oryza sativa subsp. Indica. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.

show abstract

Section: Discussionmentioning

confidence: 99%

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method

Guria

Sharma

Srikakulam

et al. 2022

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

show abstract

P-GeT Assay: An Innovative Frontier in CircRNA Enrichment

Sharma,

Guria,

Pandi

2024

Preprint

Self Cite

View full text Add to dashboard Cite

Circular RNAs have garnered attention as essential regulators of gene expression and potential biomarkers in various biological processes and diseases. However, their reliable enrichment from complex RNA pool remains a critical challenge and a costlier approach. Here, we present the "Plug-Gel Trap (P-GeT) assay," as a novel, cost-efficient, and innovative way that bypass the existing exorbitant techniques for circRNA enrichment. By greatly modifying the existing (circular DNA) gel-trap method, the principles of P-GeT assay capitalize on efficient entrapment of unique covalently-closed circular characteristics of RNAs while excluding linear counterparts. In this article, we sketch the fundamental principles behind the P-GeT assay, detailing its workflow and highlighting specificity and efficiency in aggrandizing circRNAs. We compared the competence of two regularly used gel sieves composed of either acrylamides or agarose to significantly trap circular transcripts. The selective trapping by P-GeT assay is validated using the divergent and convergent oligo-nucleotides specific to circular and linear transcripts respectively, and subsequently by northern hybridization. Both the validations are greatly able to detect only the circular form rather than linear transcript. Our study not only introduces an innovative approach for circRNA enrichment but also underscores its versatility and cost-effectiveness, making it accessible to a broader research community. The P-GeT assay represents a significant step towards advancing circRNA studies, unlocking their full potential in deciphering complex biological processes and disease mechanisms.

show abstract

Identification of Circular RNAs by Multiple Displacement Amplification and Their Involvement in Plant Development

Cited by 2 publications

References 31 publications

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method

P-GeT Assay: An Innovative Frontier in CircRNA Enrichment

Contact Info

Product

Resources

About