RNA in expression array - (Jun/28/2008 )

Hi all, I am just a student and I am gonna ask some reli naive question...

1.I have done a ChIP-ChIP, in which I quantify the DNA labelled with klenow, so that I put same amount of IP sample and genomic DNA control to hybridize one array. But for expression array, should I use same amount of cell or should I use same amount of RNA for the WT and mutant to hybridize one array? Or when should I hybridize with RNA from same amount of cell and when should I quantify the RNA and then hybridize?

2.If I break an embryo shell and isolate a kind of cell for analysis, will the expression level of cell dramatically change after isolated from their native enironment? even if they left on ice. they can change much?

Thank you for any help~~

Hi all, I am just a student and I am gonna ask some reli naive question...

1.I have done a ChIP-ChIP, in which I quantify the DNA labelled with klenow, so that I put same amount of IP sample and genomic DNA control to hybridize one array. But for expression array, should I use same amount of cell or should I use same amount of RNA for the WT and mutant to hybridize one array? Or when should I hybridize with RNA from same amount of cell and when should I quantify the RNA and then hybridize?

1. Isolate froms same amount of cells

2. Quantify and use same amount of RNA

2.If I break an embryo shell and isolate a kind of cell for analysis, will the expression level of cell dramatically change after isolated from their native enironment? even if they left on ice. they can change much?

1. It probably will change, but if you have the wt control (if you are comparing against mutant), it shouldn't matter.

2. There are some things that you can not control, so do what you can do. Leave the rest to the God of microarrays!

Thank you for any help~~