Protocol Online logo
Top : Forum Archives: : DNA Methylation, Histone and Chromatin Study

Input Control for ChIP - (Jun/12/2007 )

Hello all together,

i recently started establishing ChIP in my lab. Now I have the first results and wonder whether i unterstood the purpose of the Input control rightly.

After sonification i diluted my ChIP samples with 10 x Dilution Buffer and kept at 20 ul at -80 °C as my input control. After IP of my ChIP samples i included the input sample in the reverse crosslinking procedure. Then i performed a PCR to amplify the DNA in the input control and in the ChIP samples. Therefore i quantified the DNA in the input control and used 50 ng of DNA in the PCR. For the ChIP sample i used the same volume as for the input control. Is that correct or should i have quantified the DNA in the ChIP sample seperately and also calculated the volume corresponding to the amount.

I´m very confused!!! blink.gif blink.gif What´s the purpose of the input control? Is it just a positive control for the PCR or do i use it for quantification? How do you quantify the amount of DNA in the PCR? Do you use equal volumes or nanograms of DNA?

I´m kind of desperate . I´d appreciate your help!

-AllaboutBiochemistry-

QUOTE (AllaboutBiochemistry @ Jun 12 2007, 01:13 AM)
Hello all together,

i recently started establishing ChIP in my lab. Now I have the first results and wonder whether i unterstood the purpose of the Input control rightly.

After sonification i diluted my ChIP samples with 10 x Dilution Buffer and kept at 20 ul at -80 °C as my input control. After IP of my ChIP samples i included the input sample in the reverse crosslinking procedure. Then i performed a PCR to amplify the DNA in the input control and in the ChIP samples. Therefore i quantified the DNA in the input control and used 50 ng of DNA in the PCR. For the ChIP sample i used the same volume as for the input control. Is that correct or should i have quantified the DNA in the ChIP sample seperately and also calculated the volume corresponding to the amount.

I´m very confused!!! blink.gif blink.gif What´s the purpose of the input control? Is it just a positive control for the PCR or do i use it for quantification? How do you quantify the amount of DNA in the PCR? Do you use equal volumes or nanograms of DNA?

I´m kind of desperate . I´d appreciate your help!


Many people express their ChIP data as a ratio of PCR signal from ChIPed DNA to the input PCR signal (if you are using real time PCR an easy way to express this is 2^(Input Ct - ChIP Ct). This mostly removes the effect of differences in the efficiency of primers if you are comparing results from multiple primers.

If you are comparing several samples with the same primers this ratio may also help normalize differences in input. This normalization is only valid if your protein of interest is no where near the saturation point for your antibody. Once you start to approach saturating your antibody the response to concentration is not linear.

If you are going to calculate enrichment you should use the same concentration of DNA in both your ChIP PCR and input PCR.

If you have a negative control primer for binding (a region where you know your factor of interest doesn't bind) then there's no reason to do enrichment. Just use the same volume of your input (you may need to dilute to keep in the linear range) and ChIP DNA samples in PCR with the different primers and express your results as the PCR signal for your region of interest divided by the signal for your negative control. If you are using normal (semiquantitative) PCR and scanning your results from a gel you might try multiplexing your reactions with both negative control primer and your primer for your region of interest. Then you can take ratios of the bands from the same reaction.

Hope that helps.
Joel

-KPDE-

QUOTE (KPDE @ Jun 12 2007, 08:37 PM)
QUOTE (AllaboutBiochemistry @ Jun 12 2007, 01:13 AM)
Hello all together,

i recently started establishing ChIP in my lab. Now I have the first results and wonder whether i unterstood the purpose of the Input control rightly.

After sonification i diluted my ChIP samples with 10 x Dilution Buffer and kept at 20 ul at -80 °C as my input control. After IP of my ChIP samples i included the input sample in the reverse crosslinking procedure. Then i performed a PCR to amplify the DNA in the input control and in the ChIP samples. Therefore i quantified the DNA in the input control and used 50 ng of DNA in the PCR. For the ChIP sample i used the same volume as for the input control. Is that correct or should i have quantified the DNA in the ChIP sample seperately and also calculated the volume corresponding to the amount.

I´m very confused!!! blink.gif blink.gif What´s the purpose of the input control? Is it just a positive control for the PCR or do i use it for quantification? How do you quantify the amount of DNA in the PCR? Do you use equal volumes or nanograms of DNA?

I´m kind of desperate . I´d appreciate your help!


Many people express their ChIP data as a ratio of PCR signal from ChIPed DNA to the input PCR signal (if you are using real time PCR an easy way to express this is 2^(Input Ct - ChIP Ct). This mostly removes the effect of differences in the efficiency of primers if you are comparing results from multiple primers.

If you are comparing several samples with the same primers this ratio may also help normalize differences in input. This normalization is only valid if your protein of interest is no where near the saturation point for your antibody. Once you start to approach saturating your antibody the response to concentration is not linear.

If you are going to calculate enrichment you should use the same concentration of DNA in both your ChIP PCR and input PCR.

If you have a negative control primer for binding (a region where you know your factor of interest doesn't bind) then there's no reason to do enrichment. Just use the same volume of your input (you may need to dilute to keep in the linear range) and ChIP DNA samples in PCR with the different primers and express your results as the PCR signal for your region of interest divided by the signal for your negative control. If you are using normal (semiquantitative) PCR and scanning your results from a gel you might try multiplexing your reactions with both negative control primer and your primer for your region of interest. Then you can take ratios of the bands from the same reaction.

Hope that helps.
Joel



Thank you so much Joel.I have only one more question. rolleyes.gif rolleyes.gif rolleyes.gif You wrote, i should use the same concentration of DNA in both my ChIP PCR and input PCR to calculate enrichment. Does it mean, that after Reverse Crosslinking, i measure the DNA concentration in both ChIPped sample and input control and then calculate the corresponding volume of e.g. 50 ng?

What i have done is, that i only measured the DNA concentration in the input controls after Reverse crosslinking (for the sample later used with the target antibody and the no antibody control), calculated the volume to have the 50 ng nanograms in the PCR. When i calculated e.g. 10 ul to get 50 ng of the input control, then i used also 10 ul of ChIPped sample in the PCR. I thought, that i can normalize the amount by that, since after IP i will have less total DNA in my ChIP sample, but more enriched target DNA.

Sorry, that i act like i was born yesterday. blink.gif blink.gif

-AllaboutBiochemistry-

QUOTE (AllaboutBiochemistry @ Jun 13 2007, 03:17 AM)
QUOTE (KPDE @ Jun 12 2007, 08:37 PM)
QUOTE (AllaboutBiochemistry @ Jun 12 2007, 01:13 AM)
Hello all together,

i recently started establishing ChIP in my lab. Now I have the first results and wonder whether i unterstood the purpose of the Input control rightly.

After sonification i diluted my ChIP samples with 10 x Dilution Buffer and kept at 20 ul at -80 °C as my input control. After IP of my ChIP samples i included the input sample in the reverse crosslinking procedure. Then i performed a PCR to amplify the DNA in the input control and in the ChIP samples. Therefore i quantified the DNA in the input control and used 50 ng of DNA in the PCR. For the ChIP sample i used the same volume as for the input control. Is that correct or should i have quantified the DNA in the ChIP sample seperately and also calculated the volume corresponding to the amount.

I´m very confused!!! blink.gif blink.gif What´s the purpose of the input control? Is it just a positive control for the PCR or do i use it for quantification? How do you quantify the amount of DNA in the PCR? Do you use equal volumes or nanograms of DNA?

I´m kind of desperate . I´d appreciate your help!


Many people express their ChIP data as a ratio of PCR signal from ChIPed DNA to the input PCR signal (if you are using real time PCR an easy way to express this is 2^(Input Ct - ChIP Ct). This mostly removes the effect of differences in the efficiency of primers if you are comparing results from multiple primers.

If you are comparing several samples with the same primers this ratio may also help normalize differences in input. This normalization is only valid if your protein of interest is no where near the saturation point for your antibody. Once you start to approach saturating your antibody the response to concentration is not linear.

If you are going to calculate enrichment you should use the same concentration of DNA in both your ChIP PCR and input PCR.

If you have a negative control primer for binding (a region where you know your factor of interest doesn't bind) then there's no reason to do enrichment. Just use the same volume of your input (you may need to dilute to keep in the linear range) and ChIP DNA samples in PCR with the different primers and express your results as the PCR signal for your region of interest divided by the signal for your negative control. If you are using normal (semiquantitative) PCR and scanning your results from a gel you might try multiplexing your reactions with both negative control primer and your primer for your region of interest. Then you can take ratios of the bands from the same reaction.

Hope that helps.
Joel



Thank you so much Joel.I have only one more question. rolleyes.gif rolleyes.gif rolleyes.gif You wrote, i should use the same concentration of DNA in both my ChIP PCR and input PCR to calculate enrichment. Does it mean, that after Reverse Crosslinking, i measure the DNA concentration in both ChIPped sample and input control and then calculate the corresponding volume of e.g. 50 ng?

What i have done is, that i only measured the DNA concentration in the input controls after Reverse crosslinking (for the sample later used with the target antibody and the no antibody control), calculated the volume to have the 50 ng nanograms in the PCR. When i calculated e.g. 10 ul to get 50 ng of the input control, then i used also 10 ul of ChIPped sample in the PCR. I thought, that i can normalize the amount by that, since after IP i will have less total DNA in my ChIP sample, but more enriched target DNA.

Sorry, that i act like i was born yesterday. blink.gif blink.gif


To get true enrichment you do need to load the same amount of DNA for both your input and your ChIP PCR but in my opinion this is unnecessarilly laborious. I still think comparing your signal to a site where your factor doesn't bind is much easier.

-KPDE-