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Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.
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.
Type
Journal articleSubject
AnimalsBirds
Embryo, Nonmammalian
Gene Expression Profiling
In Situ Hybridization
RNA Probes
RNA, Complementary
Sulfur Radioisotopes
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https://hdl.handle.net/10161/9300Published Version (Please cite this version)
10.3791/3764Publication Info
Chen, CC; Wada, K; & Jarvis, Erich David (2012). Radioactive in situ hybridization for detecting diverse gene expression patterns in
tissue. J Vis Exp(62). 10.3791/3764. Retrieved from https://hdl.handle.net/10161/9300.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Erich David Jarvis
Adjunct Professor in the Deptartment of Neurobiology
Dr. Jarvis' laboratory studies the neurobiology of vocal communication. Emphasis is
placed on the molecular pathways involved in the perception and production of learned
vocalizations. They use an integrative approach that combines behavioral, anatomical,
electrophysiological and molecular biological techniques. The main animal model used
is songbirds, one of the few vertebrate groups that evolved the ability to learn vocalizations.
The generality of the discoveries is tested in other vocal lear

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