Comparative analysis of circular RNA enrichment methods

Shi, Haojie; Zhou, Ying; Jia, Erteng; Liu, Zhiyu; Pan, Min; Bai, Ya Mei; Zhao, Xiangwei; Ge, Qinyu

doi:10.1080/15476286.2021.2012632

Cited by 16 publications

(13 citation statements)

References 41 publications

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“…circRNA identification using rRNA-depleted samples yielded not only a higher quantification of transcripts, but also captured more unique transcripts than poly(A)-selected samples. These findings further prove that the RNA-seq selection protocol has a significant impact on the resulting circRNA profiles as previous studies have also observed (47), and brings to question the reliability of previous studies that have used the poly(A)-selection protocol on circRNA detection for biomarker discovery.…”

Section: Resultssupporting

confidence: 80%

Detection of circular RNAs and their potential as biomarkers predictive of drug response

Nguyen

Mammoliti

Nair

et al. 2023

Preprint

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The introduction of high-throughput sequencing technologies has allowed for comprehensive RNA species detection, both coding and non-coding, which opened new avenues for the discovery of predictive and prognostic biomarkers. However the consistency of the detection of different RNA species depends on the RNA selection protocol used for RNA-sequencing. While preliminary reports indicated that non-coding RNAs, in particular circular RNAs, constitute a rich source of biomarkers predictive of drug response, the reproducibility of this novel class of biomarkers has not been rigorously investigated. To address this issue, we assessed the inter-lab consistency of circular RNA expression in cell lines profiled in large pharmacogenomic datasets. We found that circular RNA expression quantified from rRNA-depleted RNA-seq data is stable and yields robust prognostic markers in cancer. On the other hand, quantification of the expression of circular RNA from poly(A)-selected RNA-seq data yields highly inconsistent results, calling into question results from previous studies reporting their potential as predictive biomarkers in cancer. We have also identified median expression of transcripts and transcript length as potential factors influencing the consistency of RNA detection. Our study provides a framework to quantitatively assess the stability of coding and non-coding RNA expression through the analysis of biological replicates within and across independent studies.

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Section: Resultssupporting

confidence: 80%

Detection of circular RNAs and their potential as biomarkers predictive of drug response

Nguyen

Mammoliti

Nair

et al. 2023

Preprint

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“…We found that the expression of circRNA-5335 increased continuously during the induction of differentiation, suggesting that it may be involved in the adipogenic process. Compared with gDNA, we observed that only the cDNA obtained from the RNase R-treated total RNA showed a specific band corresponding to the backsplice junction fragment [ 40 ], which verified that circRNA-5335 exists in preadipocytes as a circular molecule.…”

Section: Discussionmentioning

confidence: 89%

CircRNA-5335 Regulates the Differentiation and Proliferation of Sheep Preadipocyte via the miR-125a-3p/STAT3 Pathway

Guo,

Ciwang,

Wang

et al. 2024

Veterinary Sciences

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The content of intramuscular fat (IMF) from preadipocytes is proportional to meat quality in livestock. However, the roles of circRNAs in IMF deposition in sheep are not well known. In this study, we show that circRNA-5335/miR-125a-3p/STAT3 play a crucial adjective role in the proliferation and differentiation of sheep preadipocytes. In this study, we characterized the roles of differentially expressed circRNA-5335/miR-125a-3p/STAT3, which were screened from sheep of different months of age and based on sequencing data. Firstly, the expression profiles of circRNA-5335/miR-125a-3p/STAT3 were identified during the differentiation of preadipocytes in vitro by RT-qPCR and WB. Then, the targeting relationship of the circRNA-5335/miR-125a-3p/STAT3 was verified by dual-luciferase reporter assays. The results of RT-qPCR, CCK8, EdU and Oil Red O staining assay showed that miR-125a-3p suppressed the differentiation and raised the proliferation of preadipocytes by targeting STAT3. As a competing endogenous RNA, the downregulation of circRNA-5335 decreased the expression of STAT3 by increasing miR-125a-3p, which inhibited the differentiation of preadipocytes and promoted proliferation. Our present study demonstrates the functional significance of circRNA-5335/miR-125a-3p/STAT3 in the differentiation of sheep preadipocytes, and provides novel insights into exploring the mechanism of IMF.

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“…For example, Panda et al applied the same RTP method to enrich circular RNA in HeLa and C2C12 cells by eliminating RNase R resistant transcripts (like U1 snRNA) [ 17 , 18 ]. Shi et al analysed the differences in numbers and biotypes of identified human blood circRNAs between rRNAs removal, polyadenylation followed by RNase R treatment, and the ways that combined these two methods in different orders, respectively [ 19 ]. However, different from cells, tissues consist of specialized cells which can affect the sensitivity of these methods to enrich circRNAs.…”

Section: Discussionmentioning

confidence: 99%

Assessment of different enrichment methods revealed the optimal approach to identify bovine circRnas

Wang,

Gruninger

et al. 2024

RNA Biology

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Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal ( Ribo ); linear RNAs degradation ( R ); linear RNAs and RNAs with structured 3′ ends degradation ( RTP ); ribosomal RNAs coupled with linear RNAs elimination ( Ribo-R ); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination ( Ribo-RP ); and ribosomal RNA, linear RNAs and RNAs with structured 3′ ends elimination ( Ribo-RTP ), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads ( P adj <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.

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Comparative analysis of circular RNA enrichment methods

Cited by 16 publications

References 41 publications

Detection of circular RNAs and their potential as biomarkers predictive of drug response

Detection of circular RNAs and their potential as biomarkers predictive of drug response

CircRNA-5335 Regulates the Differentiation and Proliferation of Sheep Preadipocyte via the miR-125a-3p/STAT3 Pathway

Assessment of different enrichment methods revealed the optimal approach to identify bovine circRnas

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