We are trying to detect integration of HPV (human papillomavirus) sequences from specific HPV types in the genome of epithelial cells. The problem here, is that there are quite large similarities between the regions of the HPV that we are interested in (E6 and E7). Thus I believe that probes based on PCR products will fail to be able to distinguish between individual HPV types.
Some probes for distinguishing the different types do exists, but these tend to be targetting the L1 region, which we do not want to target....
I have also been considering using oligonucleotides to be able to target the small mismatch regions, but based on this discussion, I can understand this might be difficult...
So far I have considered using some minor amplifications by DIG-labelled probes, mouse anti-dig and fluorophore conjugated anti-mouse... But we haven't attempted it yet.
Do you guys have any ideas?
Hi,
I have never done FISH for HPV by myself but would suggest that a type-specific FISH will be very difficult- caused by the the sequence similarities. But you can type your samples first by PCR and then running the FISH for the detected HPV type. For detection of HPV integration you have to use specific protocols to eliminate episomale genomes... as published by Hopman AHN et al.
Regards,
Norman
Hi.
Thanks for the answer, and the suggestion regarding episomal genomes...
I feared as much. We are currently doing the typing with PCR followed by FISH, bu we are currently only having a limitted quantity of tissues and has to perform a large amount of different test. As such, we hoped to be able to reduce the amounts of test a little to save material..
I have managed to find some short sequences (30-40 bp) that are specific for the HPV types we are investigating, so I was wondering if it would be possible to use 30-40 bp long oligonucleotides labelled with digoxigenin and then amplify the signal through secondary antibodies - or perhaps TSA amplification... I'm, however, unsure if the fluorescence will be sufficent to be able to detect it, if only a small number of the genes of interest are pressent..
Best
Caspar
Limited quantities of clinical material is always a problem :-( but I think the idea to type samples by FISH is likely not successful. There are some protocols for oligonucleotide-based FISH analyses but I have only seen data for the detection of highly repetitive sequences. Thus it may be possible to detect multi-copy HPV integrates but problematic for single-copy genomes. If you have time you can test the method with CaSki and SiHa cells for HPV16 but you cannot control the hybridization for rare HPV types.
If you want to save material- you can try kits for combined DNA-RNA and even protein extraction. Maybe this helps.
Regards,
Norman
Thanks for the suggestion. I'll give it a try with the SiHa cells as you suggested and do some tests on cervix CIN3 / carcinoma, since we have pretty much all the material allready... You are proberly right about the more rare types of HPV...
Thanks for the good suggestions :-).
Best
Caspar
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