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In situ hybridization: identification of rare mRNAs in human tissues.

Publication ,  Journal Article
Wilson, KH; Schambra, UB; Smith, MS; Page, SO; Richardson, CD; Fremeau, RT; Schwinn, DA
Published in: Brain Res Brain Res Protoc
May 1997

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Duke Scholars

Published In

Brain Res Brain Res Protoc

DOI

ISSN

1385-299X

Publication Date

May 1997

Volume

1

Issue

2

Start / End Page

175 / 185

Location

Netherlands

Related Subject Headings

  • Spinal Cord
  • Receptors, Adrenergic, alpha
  • RNA, Messenger
  • Neurology & Neurosurgery
  • In Situ Hybridization
  • Humans
  • 5202 Biological psychology
  • 3209 Neurosciences
  • 1702 Cognitive Sciences
  • 1701 Psychology
 

Citation

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Wilson, K. H., Schambra, U. B., Smith, M. S., Page, S. O., Richardson, C. D., Fremeau, R. T., & Schwinn, D. A. (1997). In situ hybridization: identification of rare mRNAs in human tissues. Brain Res Brain Res Protoc, 1(2), 175–185. https://doi.org/10.1016/s1385-299x(96)00028-1
Wilson, K. H., U. B. Schambra, M. S. Smith, S. O. Page, C. D. Richardson, R. T. Fremeau, and D. A. Schwinn. “In situ hybridization: identification of rare mRNAs in human tissues.Brain Res Brain Res Protoc 1, no. 2 (May 1997): 175–85. https://doi.org/10.1016/s1385-299x(96)00028-1.
Wilson KH, Schambra UB, Smith MS, Page SO, Richardson CD, Fremeau RT, et al. In situ hybridization: identification of rare mRNAs in human tissues. Brain Res Brain Res Protoc. 1997 May;1(2):175–85.
Wilson, K. H., et al. “In situ hybridization: identification of rare mRNAs in human tissues.Brain Res Brain Res Protoc, vol. 1, no. 2, May 1997, pp. 175–85. Pubmed, doi:10.1016/s1385-299x(96)00028-1.
Wilson KH, Schambra UB, Smith MS, Page SO, Richardson CD, Fremeau RT, Schwinn DA. In situ hybridization: identification of rare mRNAs in human tissues. Brain Res Brain Res Protoc. 1997 May;1(2):175–185.
Journal cover image

Published In

Brain Res Brain Res Protoc

DOI

ISSN

1385-299X

Publication Date

May 1997

Volume

1

Issue

2

Start / End Page

175 / 185

Location

Netherlands

Related Subject Headings

  • Spinal Cord
  • Receptors, Adrenergic, alpha
  • RNA, Messenger
  • Neurology & Neurosurgery
  • In Situ Hybridization
  • Humans
  • 5202 Biological psychology
  • 3209 Neurosciences
  • 1702 Cognitive Sciences
  • 1701 Psychology