Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs

Chen, Dan Feng; Zhang, Lu Jun; Tan, Kezhe; Jing, Qing

doi:10.1080/13102818.2017.1398596

Cited by 34 publications

(18 citation statements)

References 27 publications

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“…Another challenge for the discovery of circRNA involves the detection and quantification of circRNA. The study of circRNAs may be hampered by template switching and rolling circle amplification during reverse transcription and by amplification bias during PCR [117,118].…”

Section: Current Limitations and Challengesmentioning

confidence: 99%

Functional role of circular RNAs in cancer development and progression

2018

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Circular RNAs (circRNAs) are a large class of endogenously expressed non-coding RNAs formed by covalently closed loops through back-splicing. High throughput sequencing technologies have identified thousands of circRNAs with high sequence conservation and cell type specific expression in eukaryotes. CircRNAs play multiple important roles in cellular physiology functioning as miRNA sponges, transcriptional regulators, RBP binding molecules, templates for protein translation, and immune regulators. In a clinical context, circRNAs expression is correlated with patient's clinicopathological features in cancers including breast, liver, gastric, colorectal, and lung cancer. Additionally, distinct properties of circRNAs, such as high stability, exonuclease resistance, and existence in body fluids, show promising role for circRNAs as molecular biomarkers for tumor diagnosis, non-invasive monitoring, prognosis, and therapeutic intervention. Therefore, it is critical to further understand the molecular mechanism underlying circRNAs interaction in tumors and the recent progress of this RNA species in cancer development. In this review, we provide a detailed description of biological functions, molecular role of circRNAs in different cancers, and its potential role as biomarkers in a clinical context.

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Section: Current Limitations and Challengesmentioning

confidence: 99%

Functional role of circular RNAs in cancer development and progression

2018

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“…Similarly, conventional reverse transcription-quantitative PCR (RT-qPCR) assays do not distinguish circular from linear RNA when using the linear genome as template for primer design. Even when actively searching for circRNAs, methodological challenges like template switching and rolling circle amplification during RT and amplification bias during PCR may hamper the results [ 1 , 23 , 24 ]. Therefore, the circular nature of the transcripts needs to be validated.…”

Section: Introductionmentioning

confidence: 99%

Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies

Dahl

Daugaard

Andersen

et al. 2018

Laboratory Investigation

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Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3′-end of an exon to the 5′-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.

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“…Our data show that the circRNAome is extensively downregulated in lesional skin. The RNA-seq data were confirmed using an enzyme-free method [36], which is not subject to PCR bias and potential artifacts related to reverse transcription [35][36][37], and could be extended to another patient cohort. In addition, we also observed an overall downregulation of circRNAs when comparing to normal healthy skin.…”

Section: Discussionmentioning

confidence: 70%

“…Template switching and rolling circle amplification during reverse-transcription (RT), as well as PCR bias, are major concerns in the circRNA research field as artifacts mimicking circRNA molecules may arise because of these phenomena [35][36][37]. We therefore employed an enzyme-free technology (termed NanoString nCounter) [36] to profile the expression of the top 50 most abundant circRNAs in the entire dataset within the same samples.…”

Section: Circrnas Are Less Abundant In Lesional Psoriatic Skin Relatimentioning

confidence: 99%

High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome

Moldovan

Okholm

Andersen

et al. 2019

Preprint

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Background: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. It is one of the most prevalent chronic inflammatory skin condition in adults worldwide, with a considerable negative impact on quality of life. Circular RNAs (circRNAs) are a recently identified type of non-coding RNA with diverse cellular functions related to their exceptional stability. In particular, some circRNAs can bind and regulate microRNAs (miRNAs), a group of RNAs that play a role in the pathogenesis of psoriasis. The aim of this study was to characterize the circRNAome in psoriasis and to assess potential correlations to miRNA expression patterns. Results:Using high-throughput RNA-sequencing (RNA-seq) and NanoString nCounter technology, we found a substantial down-regulation of circRNA expression in lesional skin compared to non-lesional skin from psoriasis patients. We saw that this mainly applies to the epidermis by analyzing laser capture microdissected tissues and by RNA chromogenic in situ hybridization (CISH). We also found that the majority of the circRNAs were downregulated independent of their corresponding linear host genes. The observed downregulation of circRNAs in psoriasis was not due to altered expression levels of factors known to affect circRNA biogenesis, nor because lesional skin contained an increased number of inflammatory cells such as lymphocytes. Finally, we saw that the overall differences in available miRNA binding sites on the circRNAs between lesional and non-lesional skin did not correlate with differences in miRNA expression patterns. Conclusions:We have performed the first genome-wide circRNA profiling of paired lesional and non-lesional skin from psoriasis patients and revealed that circRNAs are much less abundant in the lesional samples. Whether this is a cause or a consequence of the disease remains to be revealed, 3 however, we found no evidence that the loss of miRNA binding sites on the circRNAs could explain differences in miRNA expression reported between lesional and non-lesional skin. BackgroundPsoriasis is one of the most common chronic inflammatory skin conditions, with 1-3% of the adult population affected worldwide [1]. It is defined by a pronounced hyperproliferation and deficient terminal differentiation of the keratinocytes. Moreover, a complex interplay between different cell types (e.g. T cells and dendritic cells) and a variety of cytokines are known to contribute in the development of psoriasis. The pathogenesis is also based on a complex synergy between genetic predisposition, major histocompatibility alleles, and a variety of environmental triggers [2]. From the molecular point of view, however, the mechanisms responsible for the interactions between keratinocytes and inflammatory cells infiltrating the epidermis are still not fully understood. The analysis of the molecular backdrop of psoriasis has described numerous disease-associated genes and proteins with abnormal expression patterns [3], but little is...

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Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs

Cited by 34 publications

References 27 publications

Functional role of circular RNAs in cancer development and progression

Functional role of circular RNAs in cancer development and progression

Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies

High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome

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