Abstract
Simple SummaryCircular RNAs are a recently appreciated class of regulatory non-coding RNAs. Although a number of high-resolution methods have been developed for the imaging of RNAs in cells and tissues, there is no reliable method for the simultaneous imaging of circular RNAs distinctly from their linear counterparts. Here, we report circFISH as a simple and single-molecule resolution method that will simultaneously image linear and circular RNAs in fixed cells and tissues. We demonstrate that multiple circular RNAs can be imaged using circFISH. We also show the ability of circFISH to work seamlessly with protein and other organelle imaging. We optimized the method to work across sample types, making it a versatile tool for the functional characterization of circular RNAs.Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.
Highlights
A variety of regulatory RNAs have been reported to play important roles at different stages of gene expression regulation [1,2,3,4]
The idea for circFISH stems from Fusion fluorescent in situ hybridization (FISH), where we use two unique sets of probes each labeled with differently colored fluorophores to image distinct parts of chimeric fused mRNAs [33]
The second probe set, called probe linear (PL), was designed to bind exon 4, which is not a part of this circRNA (Figure 1A). The probes in this set were labeled with Texas Red. This general scheme can be used for any circRNA by choosing the exon(s) included in the circRNA to be the target region for the probe circular (PC) probe set, while the exon(s) only present in linear RNA serve as the target for the PL probe set
Summary
A variety of regulatory RNAs have been reported to play important roles at different stages of gene expression regulation [1,2,3,4]. CircRNAs have been established as key regulatory RNAs that are conserved across species and have tissue-specific expression, with their levels in the cells regulating various diseases [14]. Due to their closed-end structure, circRNAs are resistant to exonucleases, and have about a five times longer half-life than linear isoforms [15]. These circRNAs have been reported to be enriched in extracellular vesicles, highlighting their emerging role as promising biomarkers in various pathologies like neurological disorders, cardiac diseases, and many cancers [10,16]. The exonic circRNAs are the most abundant, and are reported to have the most clinical relevance [8,15,19,20,21]
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