A highly sensitive nonisotopic detection method for in situ hybridization.
In situ hybridization is a technique that allows detection of specific DNA and RNA sequences in tissue sections. Nonisotopic techniques are fast and give a precise localization of the hybridization product, but a drawback is the low sensitivity. However, the sensitivity is dependent on the detection system used. To evaluate a sensitive in situ hybridization method with nonradioactive probes we compared three different detection systems, using biotin-labeled human papillomavirus (HPV) 16 probes. The three detection systems included (i) STAV-FITC method (streptavidin-fluorescein isothiocyanate/alkaline phosphatase anti-FITC), (ii) APAAP method (mouse anti-biotin/anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase), and (iii) tyramide signal amplification (TSA) method (STAV-horseradish peroxidase (HRP)/biotinyl tyramide/STAV-HRP). The in situ hybridization methods were tested on CaSki and SiHa cells and two cervical carcinomas known to be HPV16 positive. The cells and tissues and been fixed in 4% buffered formalin and paraffin embedded. The three different detection systems gave satisfactory nuclear staining in CaSki cells (CaSki cells contain > 500 copies of HPV16 DNA) and the two cervical carcinomas. However, demonstration of HPV16 DNA in SiHa cells (SiHa cells contain one to two HPV16 genome copies) was possible only by use of the APAAP method. It was concluded that the APAAP method provides the best sensitivity among the nonisotopic detection systems and can detect single viral copies in formalin-fixed and paraffin-embedded material.
Department of Pathology, Norwegian Radium Hospital, Montebello, Oslo, Norway. Ruth.holm@labmed.uio.no
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D000818:Animals; D001710:Biotin; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods* / Q000706:statistics & numerical data; D051379:Mice; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000821:virology
D015344:DNA Probes, HPV; D004279:DNA, Viral; D001710:Biotin