Expression analysis of murine genes using in situ hybridization with radioactive and nonradioactively labeled RNA probes.
The term in situ hybridization (ISH) refers to all methods allowing the detection of specific DNA (gene loci) or RNA (gene expression products) sequences, using molecular hybridization (base pairing) of labeled nucleic acid probes to target molecules within "intact" cell populations in tissue sections or whole organisms, cultured cells, or chromosomal spreads. For more than two decades, ISH has been one of the main approaches used to characterize gene expression patterns in all laboratory animal models, especially in the context of embryonic development, as well as in human tissue or cell samples for both research and diagnostic purposes. Here, we describe several ISH protocols applied to the analysis of mouse embryos and tissues; this organism has become a reference for mammalian experimental genetics. These protocols use in vitro transcribed RNAs as probes for detection. Radiolabeled probes (using 35S as a radioisotope) allow sensitive ISH on sections of paraffin-embedded material, whereas nonradioactively (digoxigenin) labeled probes can be used both for hybridization of whole embryos (whole-mount ISH) and frozen tissue sections.
Chotteau-Lelièvre
Anne
A
Institute de Biologie de Lille, Université des Sciences et Technologies de Lille, Lille, France.
Dollé
Pascal
P
Gofflot
Françoise
F
D000818:Animals; D004076:Digoxigenin / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D005629:Frozen Sections; D015870:Gene Expression; D006652:Histological Techniques; D017403:In Situ Hybridization / Q000379:methods*; D066298:In Vitro Techniques; D051379:Mice; D016612:Paraffin Embedding; D011247:Pregnancy; D015347:RNA Probes / Q000138:chemical synthesis*; D012680:Sensitivity and Specificity
D015347:RNA Probes; D004076:Digoxigenin