2D and 3D FISH of expanded repeat RNAs in human lymphoblasts

• Published in: Methods (San Diego, Calif.) Calif.), 2017-05, Vol.120, p.49-57
• Main Authors: Urbanek, Martyna O., Michalak, Michal, Krzyzosiak, Wlodzimierz J.
• Format: Article
• Language: English
• Subjects: Amyotrophic Lateral Sclerosis - genetics; Ataxia - genetics; Cells, Cultured; Fluorescence in situ hybridization; Fluorescent Dyes - chemistry; Fragile X Syndrome - genetics; Frontotemporal Dementia - genetics; Humans; Huntington Disease - genetics; In Situ Hybridization, Fluorescence - instrumentation; In Situ Hybridization, Fluorescence - methods; Lymphocytes; Microscopy, Confocal; Molecular Imaging - instrumentation; Molecular Imaging - methods; Myotonic Dystrophy - genetics; Ribonuclear foci; RNA - chemistry; RNA - genetics; RNA live imaging; RNA toxicity; Simple repeat expansion diseases; Tremor - genetics; Trinucleotide Repeat Expansion
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2017.04.002

•Short repeat-targeting probes could image transcripts with expanded repeats.•RNA foci in simple repeat expansion diseases are found in lymphoblast cells.•3D FISH preserves cell nuclei structure and improves RNA localization analysis.•2D and 3D RNA FISH may be combined with immunofluorescence labeling.•RNA live imaging enables transcript tracking from transcription to degradation. The first methods for visualizing RNAs within cells were designed for simple imaging of specific transcripts in cells or tissues and since then significant technical advances have been made in this field. Today, high-resolution images can be obtained, enabling visualization of single transcript molecules, quantitative analyses of images, and precise localization of RNAs within cells as well as co-localization of transcripts with specific proteins or other molecules. In addition, tracking of RNA dynamics within single cell has become possible. RNA imaging techniques have been utilized for investigating the role of mutant RNAs in a number of human disorders caused by simple microsatellite expansions. These diseases include myotonic dystrophy type 1 and 2, amyotrophic lateral sclerosis/frontotemporal dementia, fragile X-associated tremor/ataxia syndrome, and Huntington’s disease. Mutant RNAs with expanded repeats tend to aggregate predominantly within cell nuclei, forming structures called RNA foci. In this study, we demonstrate methods for fluorescent visualization of RNAs in both fixed and living cells using the example of RNAs containing various expanded repeat tracts (CUG, CCUG, GGGGCC, CGG, and CAG) from experiment design to image analysis. We describe in detail 2D and 3D fluorescence in situ hybridization (FISH) protocols for imaging expanded repeats RNAs, and we review briefly live imaging techniques used to characterize RNA foci formed by mutant RNAs. These methods could be used to image the entire cellular pathway of RNAs, from transcription to degradation.