mRNA in-situ hybridization using biotinylated oligonucleotide probes: implications for the diagnostic laboratory.
It is now possible to detect low copy numbers of messenger ribonucleic acid (mRNA) while retaining good histologic morphology for the determination of specific gene expression in diseased tissues. This technology will allow the pathologist to provide important prognostic information about tumors (expression of oncogenes and growth factors), to identify the subclones within the tumor which may be most likely to metastasize (expression of adhesion molecules and proteases) and to identify etiologic genetic aberrations (viral insertions). A technique for in-situ hybridization to mRNA has been developed for use in formalin fixed paraffin embedded tissues which is suitable for a hospital histology laboratory. Optimal conditions for the procedure were determined by using a biotinylated poly (d)T oligonucleotide probe. Results were dependent on the tissue type, fixation time, condition of the tissue prior to fixation, and degree of digestion before hybridization. The temperature and conditions of hybridization were optimized so that the poly d(T) control probe and the longer test probe could be run simultaneously. Streptavidin and avidin alkaline phosphatase detection systems were tested using levamisole to minimize background staining, and a biotin blocking agent to reduce reaction to renal tubular biotin. Increasing the temperature of stringency washes did not significantly improve the specificity but had a markedly detrimental effect on tissue morphology. The mRNA appears to remain stable within routinely fixed surgical material over long periods of time allowing for large retrospective studies. A review of c-erbB-2 expression in 16 human breast lesions was carried out comparing mRNA in-situ hybridization to immunoperoxidase and cytosolic methods. By direct localization of both message and antigen, it was possible to demonstrate focal positivity that cytosolic methods did not detect. Aberrant translation was noted in one case, and c-erbB-2 expression in non-malignant breast was detected in two cases.
Szakacs
J G
JG
Department of Pathology, University of South Florida, College of Medicine, Tampa 33612.
Livingston
S K
SK
D000818:Animals; D001710:Biotin; D001943:Breast Neoplasms / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005348:Fibrocystic Breast Disease / Q000378:metabolism; D018734:Genes, erbB-2; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013194:Staining and Labeling
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D001710:Biotin