Hello All,
I am studying epigenetic modifications in children with reference to disease condition. I have to analyse three histone modifications at the same time in a single blood sample by performing Chromatin Immunoprecipitation assay (ChIP). For each modification I have to use separate antibody and at the end of the reaction I have to do Real Time PCR assay (ChIP-PCR). I have a doubt on sample preparation part.
My question is how can I prepare chromatin fraction to analyse those three modifications?
1. First should I have to isolate PBMC and separate chromatin complex (with DNA) and do Immunoprecipitation with three specific antibodies at the same time and at last purify DNA for PCR reaction? Will it work efficiently in this way?
2. Second should I have to prepare chromatin complex and aliquot it equally into three, each for separate ChIP-PCR reaction? Will it work efficiently in this way?
Hi Kathirvel, you should go for the second option. If you add all three antibodies at the same time, it will be impossible (or at least be extremely complicated) to separate your DNA afterwards. In your first option, you will only be able to determine if a DNA fragment is covered by one or more of your modifications, but not which.
Keep in mind that you will need quite some cells to get a good ChIP, especially if the efficiency of your antibody is not optimal. Plan and optimize your experiment well.
I completely agree with Daan. You can only assess one histone PTMs at one time using standard ChIP approach. What you are suggesting has been in everybody mind from the field for longtime but the technique is not public yet…the way to turn around for up to now is to perform sequential ChIP or Re-ChIP….That is the ChIPped material from the first ChIP is subjected to a second ChIP with another antibody, etc…
You can choose between native or formaldehyde crosslink. Try to use micro ChIP if you have very little cells to work with and just follow the published protocol.
I really thank you for your answers. It was helpful for me....And one more thing i need to get clarified is, I am planning to store the extracted chromatin for a long time say around 1 and Half years...because of the rarity of my disease prevalance....If I do the same efficiency will i get it? and if i do repeated ChIP the efficiency will be there? I have gone through the literatures but i am unable to get a clear picture. Could you peolpe guide me in this regard?
Extended storage will always have some effect on your samples, but if you store your sample consistently at -80c the effect will most likely be negligible. Repeated freezing and thawing should be avoided at all cost, so aliquot your samples before freezing if you expect to do your final experiments at different times.
You can follow the protocol described in my publication "Epigenetic profile of the euchromatic region of human Y chromosome." Nucleic Acids Research 01/2011; 39(9):3594-606..
Hello,
I am also doing the histone modifications using ChIP analysis. I have some basic query. I want to do ChIP analysis for histone methylation followed by qPCR related to a particular gene. So, is it possible to study histone methylation in relation to a particular gene? If yes, then at which stage should we use particular primers for that? If it is, are primers specific for only the promoter region of that gene? kindly guide me as soon as possible.
@Neerja Aggarwal,
Yeah, U can do it. First U have to isolate the chromatin pack along with bounded DNA and then go for histone modification by CHIP analysis. The same can be used later for DNA isolation and can be used as a DNA source for UR DNA methylation studies. There is a fact that U must have known, higher the methylation level at promoter region, lower the gene expression. Say it's Inversely Proportional. So, design a primer specific to CpG island and do PCR....
All the best...
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