Which markers of epigenetic state can be measured by RT PCR?

[max_rempel]

I need good advice. I am doing a bunch of RT PCRs from mouse skin samples with Taqman. This takes all my technical capacity and I can not do any DNA methylation analyses at the moment. The question: how to characterize the overal epigenetic state of keratinocytes in the skin (after different drug and UV treatments) using RT PCR?
I realize that the answers will be incomplete and this is not a typical way of doing epigenetics, but I do want to do few relevant genes and I need help in picking them.

I am thinking about choosing between DNMTs, dicer, siRNA pathway genes, histones, possibly some histone-modifying enzymes... But I am not sure which of them are actually regulated on transcriptional level and how the level of their expression would correlate with global DNA silencing in the cell. The hypothesis is that UV and drugs are affecting the epigenetic status of keratinocytes and they become more or less prone to carcinogenic transformation (this part is covered in UV-carcinogenesis protocol which ends up with real tumors, but the early epigenetic changes leading to cancer - that is what I need to measure).

[admin]

Hello Max,
I am not an expert at epigenetics, although the guy who wrote many of the articles for this page and site did research on epigenetics for several years.
Epigenetics Methylation Station

RTPCR was used by him here:
Reverse transcriptase polymerase chain reaction
I would also check out many papers on pubmed to see what others are doing with RT-PCR. Can you use other methods as well?

You can check out the protocols and methods used here
Techniques Study Epigenetics
But you can use RT-PCR to check protein levels although the cells are treated or the cell lines are with AZA or other chemicals, as you will be using keratinocytes you can do this.

Please let us know what you end up doing, I would like to learn too

Cheers

[max_rempel]

Thanks for the links.
I will try also to measure global methylcytosine by HPLC and possibly methylation of SINEs and LINEs by restriction-PCR.
If you know any good combination of primers and restriction enzyme for mice, I would appreciate that info. Otherwise I am thinking about designing the assay from scratch.

In any case, I am running Taqmans now and adding few genes of interest would be easiest.