Determination of gene expression patterns using in situ hybridization to Drosophila testes

Morris, Ceri, Benson, Elizabeth and White-Cooper, Helen ORCID: https://orcid.org/0000-0002-3373-8023 2009. Determination of gene expression patterns using in situ hybridization to Drosophila testes. Nature Protocols 4 (12) , pp. 1807-1819. 10.1038/nprot.2009.192

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Abstract

We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2–48 samples, and takes 4–5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QH Natural history > QH426 Genetics
Uncontrolled Keywords:	Genetic analysis; Model organisms; Nucleic acid based molecular biology
Publisher:	MacMillan
ISSN:	1754-2189
Last Modified:	04 Jan 2023 02:05
URI:	https://orca.cardiff.ac.uk/id/eprint/8855

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