Imaging of Telomerase RNA by Single-Molecule Inexpensive FISH Combined with Immunofluorescence

• Published in: STAR protocols Vol. 1; no. 2; p. 100104
• Main Authors: Querido, Emmanuelle, Sfeir, Agnel, Chartrand, Pascal
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.09.2020; Elsevier
• Subjects: Coiled Bodies - metabolism; Fluorescent Antibody Technique - methods; HeLa Cells; Humans; In Situ Hybridization, Fluorescence - methods; Protocol; RNA - chemistry; RNA - genetics; Single Molecule Imaging - methods; Telomerase - chemistry; Telomerase - genetics; Telomere - metabolism
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100104

Fluorescent in situ hybridization (FISH) on the RNA moiety of human telomerase (hTR) with 50-mer probes detects hTR RNA accumulated in Cajal bodies. Using both live-cell imaging and single-molecule inexpensive FISH, our published work revealed that only a fraction of hTR localizes to Cajal bodies, with the majority of hTR molecules distributed throughout the nucleoplasm. This protocol is an application guide to the smiFISH method for the dual detection of hTR RNA and telomeres or Cajal bodies by immunofluorescence. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020). [Display omitted] •RNA smiFISH with multiple small probes reveals single molecules of hTR RNA in nucleus•The smiFISH technique is compatible with immunofluorescence for colocalization assay•Colocalized regions can be mapped in 3D images with the open source 3D ImageJ Suite Fluorescent in situ hybridization (FISH) on the RNA moiety of human telomerase (hTR) with 50-mer probes detects hTR RNA accumulated in Cajal bodies. Using both live-cell imaging and single-molecule inexpensive FISH, our published work revealed that only a fraction of hTR localizes to Cajal bodies, with the majority of hTR molecules distributed throughout the nucleoplasm. This protocol is an application guide to the smiFISH method for the dual detection of hTR RNA and telomeres or Cajal bodies by immunofluorescence.