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Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.

dc.contributor.author Chen, CC
dc.contributor.author Jarvis, Erich David
dc.contributor.author Wada, K
dc.coverage.spatial United States
dc.date.accessioned 2014-12-15T16:39:37Z
dc.date.issued 2012-04-27
dc.identifier http://www.ncbi.nlm.nih.gov/pubmed/22566072
dc.identifier 3764
dc.identifier.uri https://hdl.handle.net/10161/9300
dc.description.abstract Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.
dc.language eng
dc.relation.ispartof J Vis Exp
dc.relation.isversionof 10.3791/3764
dc.subject Animals
dc.subject Birds
dc.subject Embryo, Nonmammalian
dc.subject Gene Expression Profiling
dc.subject In Situ Hybridization
dc.subject RNA Probes
dc.subject RNA, Complementary
dc.subject Sulfur Radioisotopes
dc.title Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.
dc.type Journal article
pubs.author-url http://www.ncbi.nlm.nih.gov/pubmed/22566072
pubs.issue 62
pubs.organisational-group Basic Science Departments
pubs.organisational-group Duke
pubs.organisational-group Duke Institute for Brain Sciences
pubs.organisational-group Institutes and Provost's Academic Units
pubs.organisational-group Neurobiology
pubs.organisational-group School of Medicine
pubs.organisational-group University Institutes and Centers
pubs.publication-status Published online
dc.identifier.eissn 1940-087X


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