Abstract
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (In Situ) or in the entire tissue (whole mount ISH). Localization of endogenous transcripts is a desirable approach for confirming expression patterns. This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. However, RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. RNA–RNA hybrids approach may offer increased sensitivity, which is more stable than that of DNA–RNA hybrids. Here we describe the efficient ISH protocol for nonradioactive (i.e., in direct methods using digoxigenin (DIG) system) RNA probes, and it can be performed in less than 3 days.
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References
Mary LP, Gall JG (1969) Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc Natl Acad Sci USA 64:600–604
John HA, Birnstiel ML et al (1969) RNA-DNA hybrids at the cytological level. Nature 223:582–587
Olivier B, Walter W (1998) A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1:10–16
Singer RH, Ward DC (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci USA 79:7331–7335
Farquharson M, Harvie R, McNicol AM (1990) Detection of messenger RNA using a digoxigenin end-labeled oligonucleotide probe. J Clin Pathol 43:423–428
Morris RG, Arends MJ, Bishop PE et al (1990) Sensitivity of digoxigenin and biotin-labeled probes for detection of human papillomavirus by in situ hybridization. J Clin Pathol 43: 800–805
Science RA (2008) DIG application manual for nonradioactive in situ hybridization, 4th edn. Roche Diagnostics GmbH, Mannheim, Germany
Sugimoto N, Nakano S, Katoh M et al (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34:11211–11216
Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Valentino KL, Eberwine JH, Barchas JD (1987) In situ hybridization: applications to neurobiology. Oxford University Press, Oxford
Naber S, Smith L, Wolfe HJ (1992) Role of the frozen tissue bank in molecular pathology. Diagn Mol Pathol 1:73–79
Herrington CS, McGee JO (1992) Principles and basic methodology of DNA/RNA detection by in situ hybridization. In: Herrington CS, McGee JO (eds) Diagnostic molecular pathology: a practical approach, vol 1. IRL, New York, pp 69–102
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Lee, D., Xiong, S., Xiong, WC. (2013). General Introduction to In Situ Hybridization Protocol Using Nonradioactively Labeled Probes to Detect mRNAs on Tissue Sections. In: Zhou, R., Mei, L. (eds) Neural Development. Methods in Molecular Biology, vol 1018. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-444-9_16
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DOI: https://doi.org/10.1007/978-1-62703-444-9_16
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-443-2
Online ISBN: 978-1-62703-444-9
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