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  • CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs.

CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs.

Cancers (2022-01-22)
Aakash Koppula, Ahmed Abdelgawad, Jlenia Guarnerio, Mona Batish, Vijay Parashar
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

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Monoclonal Anti-AGO2 antibody produced in rat, ~1.5 mg/mL, clone 11A9, purified immunoglobulin
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Goat Anti-Rat IgG Antibody, FITC conjugate, Species Adsorbed, Chemicon®, from goat
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Dextran sulfate sodium salt from Leuconostoc spp., for molecular biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer
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DAPI, for nucleic acid staining
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Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma