Simultaneous detection of nuclear and cytoplasmic RNA variants utilizing Stellaris® RNA fluorescence in situ hybridization in adherent cells.
Methods Mol Biol
; 1211: 189-99, 2014.
Article
em En
| MEDLINE
| ID: mdl-25218386
ABSTRACT
RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool for the detection and localization of RNA and is increasingly becoming an important complement to other gene expression analysis methods. We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple RNA gene products and RNA variants in fixed mammalian cells. The technique utilizes fluorescently pre-labeled, short DNA oligonucleotides (20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in punctate fluorescent signals representing individual RNA molecules without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the quantification of single RNA transcripts. Additionally, by utilizing probe sets with spectrally distinct fluorophores, multiplex analysis of specific RNAs, or RNA variants, can be achieved. We focus on the detection of a cytoplasmic mRNA and a nuclear long noncoding RNA to illustrate the benefits of this method for cell-specific detection and subcellular localization. Post-processing of images and spot counting is briefly discussed to demonstrate the capabilities of this method for the statistical analysis of RNA molecule number per cell, which is information that can be utilized to determine overall gene expression levels and cell-to-cell gene expression variation.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
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Núcleo Celular
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Hibridização in Situ Fluorescente
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Citoplasma
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Inibidor de Quinase Dependente de Ciclina p21
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RNA Longo não Codificante
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article