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Improvement of Non-radioactive in situ Hybridization in Human Airway Tissues: Use of PCR-generated Templates for Synthesis of Probes and an Antibody Sandwich in Technique for Detection of Hybridization

Citation

Divjak, M and Glare, EM and Walters, EH, Improvement of Non-radioactive in situ Hybridization in Human Airway Tissues: Use of PCR-generated Templates for Synthesis of Probes and an Antibody Sandwich in Technique for Detection of Hybridization, Journal of Histochemistry and Cytochemistry, 50, (4) pp. 541-548. ISSN 0022-1554 (2002) [Refereed Article]

DOI: doi:10.1177/002215540205000411

Abstract

We describe the use of non-traditional methods of probe synthesis and quantification and detection of hybridization that appreciably improved non-radioactive in situ hybridization (ISH) in human airway tissue. To avoid the problems of bacterial cloning, plasmid digestion, and probe hydrolysis, we synthesised complementary RNA probes (riboprobes) for ISH from PCR-generated DNA. DNA template was produced by nested PCR incorporation of T7 and SP6 RNA polymerase promoters. We then compared the efficiency of in vitro transcription from PCR-generated template with traditional plasmid template by quantifying the relative probe fluorescence in denaturing gels. Transcription with SP6 or T7 polymerase in either orientation produced TNF riboprobes from a single PCR-generated template more efficiently than from plasmid, providing there were no primer hairpin loops. Fluorescence quantification enabled equal amounts of probe label to be used in ISH, eliminating signals from the sense probe and demonstrating that probes transcribed from PCR templates were as sensitive as hydrolyzed probe transcribed from plasmid. Detection of ISH by a conventional anti-hapten, alkaline phosphatase-based technique was found to cause tissue damage due to extended substrate incubation at high pH. We therefore developed a four-layer, avidin-biotin-peroxidase technique that afforded greater sensitivity, allowing brief substrate incubation and resulting in structural preservation of tissue.

Item Details

Item Type:Refereed Article
Research Division:Medical and Health Sciences
Research Group:Cardiorespiratory Medicine and Haematology
Research Field:Respiratory Diseases
Objective Division:Health
Objective Group:Clinical Health (Organs, Diseases and Abnormal Conditions)
Objective Field:Respiratory System and Diseases (incl. Asthma)
Author:Walters, EH (Professor Haydn Walters)
ID Code:48934
Year Published:2002
Web of Science® Times Cited:16
Deposited By:Menzies Institute for Medical Research
Deposited On:2007-08-01
Last Modified:2011-11-29
Downloads:0

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