Abstract
RNA plays a critical role in the health/maintenance of cells and misregulation of RNA contributes to the development of many disorders. Fluorescence in situ hybridization (FISH) is a useful tool for detecting the location of RNA and its targeting in intact cellular and tissue environment. The combination of FISH and immunofluorescence staining (IFS) presents a powerful method for visualizing spatial relationships or interactions between mRNA and proteins, or for localizing mRNA in certain cell types, while preserving the anatomical structure of the cell or tissue. Although seemingly straightforward, FISH/IFS of mRNA and proteins can be difficult to perform simultaneously on the same specimen often generating variable results. Here, we describe a combined method of multicolor FISH/IFS and explore various factors that influence the outcomes of protein and mRNA detection in detail.
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References
Mortimer SA, Kidwell MA, Doudna JA (2014) Insights into RNA structure and function from genome-wide studies. Nat Rev Genet 15:469–479
Ibrahim F, Nakaya T, Mourelatos Z (2012) RNA dysregulation in diseases of motor neurons. Annu Rev Pathol 7:323–352
Li Y, Wang X (2016) Role of long noncoding RNAs in malignant disease. Mol Med Rep 13:1463–1469
Wang J, Chen J, Sen S (2016) MicroRNA as biomarkers and diagnostics. J Cell Physiol 231:25–30
Mansoori B, Sandoghchian Shotorbani S, Baradaran B (2014) RNA interference and its role in cancer therapy. Adv Pharm Bull 4:313–321
Ma B, Savas JN, Chao MV et al (2012) Quantitative analysis of BDNF/TrkB protein and mRNA in cortical and striatal neurons using α-tubulin as a normalization factor. Cytometry A 81:704–717
Ma B, Savas JN, Yu MS et al (2011) Huntingtin facilitates β-actin mRNA transport in dendrites of cortical neurons. Sci Rep 1:140
Zimmerman SG, Peters NC, Altaras AE et al (2013) Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat Protoc 8:2158–2179
Kwon S (2013) Single-molecule fluorescence in situ hybridization: quantitative imaging of single RNA molecules. BMB Rep 46:65–72
Shaffer SM, Wu MT, Levesque MJ et al (2013) Turbo FISH: a method for rapid single molecule RNA FISH. PLoS One 8, e75120
Renwick N, Cekan P, Masry PA et al (2013) Multicolor microRNA FISH effectively differentiates tumor types. J Clin Invest 123:2694–2702
Lea R, Bonev B, Dubaissi E et al (2012) Multicolor fluorescent in situ mRNA hybridization (FISH) on whole mounts and sections. Methods Mol Biol 917:431–444
Kochan J, Wawro M, Kasza A (2015) Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH. Biotechniques 59:209–212
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Ma, B., Tanese, N. (2017). RNA-Directed FISH and Immunostaining. In: Liehr, T. (eds) Fluorescence In Situ Hybridization (FISH). Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-52959-1_34
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DOI: https://doi.org/10.1007/978-3-662-52959-1_34
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Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-662-52957-7
Online ISBN: 978-3-662-52959-1
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