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

Publication ,  Journal Article
Chen, C-C; Wada, K; Jarvis, ED
Published in: J Vis Exp
April 27, 2012

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.

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Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

April 27, 2012

Issue

62

Location

United States

Related Subject Headings

  • Sulfur Radioisotopes
  • RNA, Complementary
  • RNA Probes
  • In Situ Hybridization
  • Gene Expression Profiling
  • Embryo, Nonmammalian
  • Birds
  • Animals
  • 3101 Biochemistry and cell biology
  • 1702 Cognitive Sciences
 

Citation

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Chen, C.-C., Wada, K., & Jarvis, E. D. (2012). Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue. J Vis Exp, (62). https://doi.org/10.3791/3764
Chen, Chun-Chun, Kazuhiro Wada, and Erich D. Jarvis. “Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.J Vis Exp, no. 62 (April 27, 2012). https://doi.org/10.3791/3764.
Chen, Chun-Chun, et al. “Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.J Vis Exp, no. 62, Apr. 2012. Pubmed, doi:10.3791/3764.

Published In

J Vis Exp

DOI

EISSN

1940-087X

Publication Date

April 27, 2012

Issue

62

Location

United States

Related Subject Headings

  • Sulfur Radioisotopes
  • RNA, Complementary
  • RNA Probes
  • In Situ Hybridization
  • Gene Expression Profiling
  • Embryo, Nonmammalian
  • Birds
  • Animals
  • 3101 Biochemistry and cell biology
  • 1702 Cognitive Sciences