Negative Control for ChIP realtime PCR in Mouse - (Feb/05/2009 )

I need a good negative control for my ChIP experiment to show my mentor that the changes I found with treatment is not an artifact

So I need a pair of primers that amplifies a region in the MOUSE genome that has no H3K4 trimethylation

Couldn't see any in PubMed....

Any suggestions will be welcomed.

Thanks

jiro_killua on Feb 5 2009, 10:55 AM said:

H3K4 trimethylation is a mark of any actively transcribed gene that has polII binding.

So, any gene that is silent in the cells/tissue of your interest will be a good negative control.

I would run away from all ubiquitously expressed genes (beta-actin, gapdh, laminb1 etc). Look for something like neurone-specific gene if you are working on erythroid cells and so on so forth.

cellcounter on Feb 5 2009, 11:14 AM said:

That sounds good

Without thinking, I ordered primers in the Gapdh gene and see the same changes with treatment

We totally freak out, wondering if our findings in the other target genes were artifacts!

jiro_killua on Feb 5 2009, 10:36 AM said:

You haven't wasted your time then! You actually proved that your technique works! Use the data for Gapdh as a positive control. Will be accepted everywhere.

Unless you are absolutely sure that the negative control gene that you chose is absolutely not expressed in your cells, I would do two different genes as negative control, just in case one that you think is not expressed is in reality an expressed gene.

Caution**

If you are treating the cells with some compound that is supposed to specifically induce or repress your gene, then Gapdh would still be a good negative control. In fact, the best negative control.

Because then you are comparing between gene expression levels before and after the treatment. And not between two genes in a static manner.

Gapdh should not be changing if your treatment is gene-promoter specific, it should change only if the compound treatment has a global affect on histone methylation levels.

cellcounter on Feb 5 2009, 12:24 PM said:

The fact is we first saw changes in some of the genes we are interested

But we don't know whether that's caused by a global change in H3K4, or if that's very specific change only at some genes

Maybe I should to a western to see if there's global difference,

But what do you think about the Gapdh findings?
Specific changes in certain genes?
Global Changes in whole genome?
Artifact: difference between samples?

jiro_killua on Feb 5 2009, 12:41 PM said:

With as much as I know about your project (gene, treatment, cells, experimental plan, your technical hand & final goal)
: All of the above!
Doing western: also depends on what you ultimately want to prove.

cellcounter on Feb 5 2009, 11:14 AM said:

So what non-expressing gene would be good if I'm working on liver, kidney and lung?

I think you can choose the neagative region in coding sequence , intron or exon can be selaected, for the acelation modification is in the promoter region NOT in RNA sequence.

jiro_killua on Feb 5 2009, 02:39 PM said:

jiro_killua on Feb 5 2009, 02:39 PM said:

Shouldn't be any rhodopsin expression in those tissues. I think that would make a great negative control. Maybe b-globin as well but you have to look out with that one. Some primers may amplify several other globins as well which can screw up your quantitation. You just have to check your candidate primers with virtual PCR first.