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Negative Control for ChIP realtime PCR in Mouse


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#1 jiro_killua

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Posted 05 February 2009 - 08:55 AM

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

#2 cellcounter

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Posted 05 February 2009 - 09:14 AM

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

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.

#3 jiro_killua

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Posted 05 February 2009 - 09:36 AM

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

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.



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!

#4 cellcounter

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Posted 05 February 2009 - 09:48 AM

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

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.



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!

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.

#5 cellcounter

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Posted 05 February 2009 - 10:24 AM

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.

#6 jiro_killua

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Posted 05 February 2009 - 10:41 AM

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.



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?

#7 cellcounter

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Posted 05 February 2009 - 11:25 AM

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.



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?

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.

#8 jiro_killua

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Posted 05 February 2009 - 01:39 PM

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

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.



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

#9 seahow

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Posted 06 February 2009 - 07:32 PM

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.

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

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.



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



#10 KPDE

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Posted 07 February 2009 - 07:56 PM

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

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.



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


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.

#11 TanyHark

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Posted 08 February 2009 - 09:10 AM

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

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.



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


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.

Rhodopsin sounds good. Nah to b-globin. All tissues have RBCs in their blood vessels and many of them could be nucleated depending upon the age or treatment of the mice. So avoid using as negative control genes expressed in blood cells , that includes WBC and platelets. Same goes with genes expressed in blood vessel wall (endothelial) too.

#12 jiro_killua

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Posted 13 February 2009 - 11:42 AM

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

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.



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


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.

Rhodopsin sounds good. Nah to b-globin. All tissues have RBCs in their blood vessels and many of them could be nucleated depending upon the age or treatment of the mice. So avoid using as negative control genes expressed in blood cells , that includes WBC and platelets. Same goes with genes expressed in blood vessel wall (endothelial) too.



If I picked a non-expressing gene, and see the % Input is really low, but there's still a difference between control and treatment, what does that mean?

like 0.0001% vs 0.00003%

If I then normalize the data using this "looks-like-noise", the difference between my real samples might be gone...

What's the interpretation?

Edited by jiro_killua, 13 February 2009 - 11:43 AM.


#13 nleboarder

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Posted 25 March 2013 - 03:13 PM

I am having a similiar problem. I am using 2 cell types, fibroblasts and a B cell tumor cell line. I am doing ChIP for H3, H3K27me3 and H3K4me3. The validation works perfectly in all of my fibroblast samples, but completely fails in all of my B cell samples! For validiation for H3 I am using RPL30, for H3K27me3, MyoD1, and for H3K4me3, Hoxc11. Why does it work great for fibroblasts and not B cells?

Also, I have read that GapDH is considered a positive control for H3K4me3. I used it as my negative for enrichment and it works really nicely. In fact, when I am using my fibroblast cells, if I compare enrichment between GapDH and Hoxc11, I get on average around a 200 fold difference! (but essentially NO difference in B cells....errrrr)

Anyone out there have an issue validating in multiple cell lines?




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