Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridisation.
In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffin sections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution.
Burns
J
J
University of Oxford, Nuffield Department of Pathology, John Radcliffe Hospital.
Graham
A K
AK
Frank
C
C
Fleming
K A
KA
Evans
M F
MF
McGee
J O
JO
D002584:Cervix Uteri / Q000382:microbiology*; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D014014:Tissue Adhesives; D014412:Tumor Virus Infections / Q000382:microbiology*
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D014014:Tissue Adhesives