FISH - whole chromosome (Apr/22/2006 )

Hi all,

I'm writing a grant proposal for PhD candidacy exam. The thing is I have to prepare a research that has nothing to do with my thesis...

My question is: I need help from an experience scientist who did a FISH analysis for the whole chromosome, I want to know what is the potential difficulties with doing this kind of procedure and how to overcome this difficulties or at least give me a good reference that I can refer to..

Thanks..

what would you be trying to find out with whole chromosome FISH? it is relatively straightforward depending on your application.

Nick

Thanks for the reply Nick,

I'm trying to find whether the gene I'm invistigating had escape X inacivation?

So I'm proposing to do RNA in situ hybridization for the XIST gene and the gene of intrest...

So I will have 3 signals, one for the XIST gene on the inactive X and 2 same signals of the gene one on the active and another one on the inactive X chromosome as identified from the XIST gene signal location...

Sicne I did not actualy did FISH before, I'm asking is this a good way of answering my proposed hypothesis and is their any difficulties or points I should keep in maind in preforming this kind of assays??

Thanks

what an interesting experiment! please let me know how you go.

Firstly, are you planning to perform FISH on interphase cells or metaphase cells? with interphase, without confocal, it would be challenging to determine which signals your gene of interest is on which chromosome. Metaphase would be the way to go, but there is a caveat, does your gene express during metaphase? the answer is probably no because the chromatin has condensed to a compact form that gives rise to the chromosome, so to perform RNA FISH to determine expression would not work because it may not be expressing at metaphase!!!

There is another way possibly to determine X-inactivation status and this would involve combined immunofluorescene and then FISH using a antibody against acetylated histone H3, the inactive X would not stain for this antibody.

However you would need to resolve the issue of expression during the cell cycle to be able to see your transcript!

I haven't performed RNA FISH before but am aware of this link which might be of some help
RNA FISH protocol