2D and 3D FISH of expanded repeat RNAs in human lymphoblasts
PBN-AR
Instytucja
Instytut Chemii Bioorganicznej Polskiej Akademii Nauk
Informacje podstawowe
Główny język publikacji
en
Czasopismo
METHODS (35pkt w roku publikacji)
ISSN
1046-2023
EISSN
1095-9130
Wydawca
ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA
DOI
Rok publikacji
2017
Numer zeszytu
Strony od-do
49-57
Numer tomu
120
Identyfikator DOI
Liczba arkuszy
Słowa kluczowe
en
Fluorescence in situ hybridization
RNA live imaging
Simple repeat expansion diseases
RNA toxicity
Ribonuclear foci
Streszczenia
Język
en
Treść
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.
Inne
System-identifier
PX-59707105d5de27874a873ec2
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