# QPCR data anlaysis - (Oct/03/2008 )

Please, guys, I need someone to help me to analyze the real time data of the ChIP experiment.
The data I have is Ct from the IP, Ct from Input, Ct from IgG. I dont know how to express the data... or better, I know how I want to express the data but I dont no how mathematically do it. I want to consider the Ct of the IgG.
Does anyone have a Ct value of the IP bigger than the Ct from the input?

-nati-

QUOTE (nati @ Oct 3 2008, 07:06 AM)
Please, guys, I need someone to help me to analyze the real time data of the ChIP experiment.
The data I have is Ct from the IP, Ct from Input, Ct from IgG. I dont know how to express the data... or better, I know how I want to express the data but I dont no how mathematically do it. I want to consider the Ct of the IgG.
Does anyone have a Ct value of the IP bigger than the Ct from the input?

Hi,

I am kind of new to this field too and have a similar question, which might help the discussion and so I bring it up here.

say, you are comparing control vs treatment:
using the primer for you target,

CT values (for simplicity):
Control Input: 20
Control Target: 30
Control IgG: 40

Treatment Input: 20
Treatment Target: 28
Treatment IgG: 40

so you will conclude that treatment cause 2 cycles of enrichment (4-fold increase)

OK, now my question:
1. If the Treatment IgG Ct=38, what will you say?
2. Is this comparison really valid? Since we don't have a control to normalize each sample (like 18S), errors in processing the sample will affect the Ct (for example, after phenol-chloroform extraction, in one sample you are able to take 480ul of the aqueous, and in another sample, you can only take 400ul, wouldn't that affect the final Ct?)

Hope someone can answer these questions, thanks a lot!!

-jiro_killua-

QUOTE (jiro_killua @ Oct 22 2008, 07:27 AM)
QUOTE (nati @ Oct 3 2008, 07:06 AM)
Please, guys, I need someone to help me to analyze the real time data of the ChIP experiment.
The data I have is Ct from the IP, Ct from Input, Ct from IgG. I dont know how to express the data... or better, I know how I want to express the data but I dont no how mathematically do it. I want to consider the Ct of the IgG.
Does anyone have a Ct value of the IP bigger than the Ct from the input?

Hi,

I am kind of new to this field too and have a similar question, which might help the discussion and so I bring it up here.

say, you are comparing control vs treatment:
using the primer for you target,

CT values (for simplicity):
Control Input: 20
Control Target: 30
Control IgG: 40

Treatment Input: 20
Treatment Target: 28
Treatment IgG: 40

so you will conclude that treatment cause 2 cycles of enrichment (4-fold increase)

OK, now my question:
1. If the Treatment IgG Ct=38, what will you say?
2. Is this comparison really valid? Since we don't have a control to normalize each sample (like 18S), errors in processing the sample will affect the Ct (for example, after phenol-chloroform extraction, in one sample you are able to take 480ul of the aqueous, and in another sample, you can only take 400ul, wouldn't that affect the final Ct?)

Hope someone can answer these questions, thanks a lot!!

Well, kind of giving my own question a second thought,

probably comparing Ct values are not very appropriate....

Think about the first question, using the same set of data:

Control (Input: 20; Target: 30; IgG: 40)
Treatment (Input: 20; Target: 28; IgG: 38)

If I do a standard curve, I will probably see this:

Control (Input: 500ug; Target: 2ug; IgG: 0.001ug)
Treatment (Input: 500ug; Target: 8ug; IgG: 0.004ug)

So there's definitely enrichment,
as Control Target / Input = 2ug/500ug=0.4%
vs Treatment Target / Input = 8ug/500ug=1.6%

And comparing the IgG, Ct of 38 vs 40 seems like a 4-fold difference, but when converted to absolute values, 0.001ug and 0.004ug (compared with 2ug and 8ug) are both negligable....

Of course, I made up all these numbers, but conceptually, I think now I understand, if the Ct value of IgG is much larger than your target, there's no reason to worry about~

-jiro_killua-

Hi, i am new in doing MeDIP/ CHIP followed by Realtime PCR .
I saw u did same stuffs. Would u mind to share the protocol and the problems you faced regarding specificity of CHIP, and did u make std curve? if yes, then is it with serial dilutions of DNA from another cells?

thanks.

QUOTE (jiro_killua @ Oct 22 2008, 11:31 AM)
QUOTE (jiro_killua @ Oct 22 2008, 07:27 AM)
QUOTE (nati @ Oct 3 2008, 07:06 AM)
Please, guys, I need someone to help me to analyze the real time data of the ChIP experiment.
The data I have is Ct from the IP, Ct from Input, Ct from IgG. I dont know how to express the data... or better, I know how I want to express the data but I dont no how mathematically do it. I want to consider the Ct of the IgG.
Does anyone have a Ct value of the IP bigger than the Ct from the input?

Hi,

I am kind of new to this field too and have a similar question, which might help the discussion and so I bring it up here.

say, you are comparing control vs treatment:
using the primer for you target,

CT values (for simplicity):
Control Input: 20
Control Target: 30
Control IgG: 40

Treatment Input: 20
Treatment Target: 28
Treatment IgG: 40

so you will conclude that treatment cause 2 cycles of enrichment (4-fold increase)

OK, now my question:
1. If the Treatment IgG Ct=38, what will you say?
2. Is this comparison really valid? Since we don't have a control to normalize each sample (like 18S), errors in processing the sample will affect the Ct (for example, after phenol-chloroform extraction, in one sample you are able to take 480ul of the aqueous, and in another sample, you can only take 400ul, wouldn't that affect the final Ct?)

Hope someone can answer these questions, thanks a lot!!

Well, kind of giving my own question a second thought,

probably comparing Ct values are not very appropriate....

Think about the first question, using the same set of data:

Control (Input: 20; Target: 30; IgG: 40)
Treatment (Input: 20; Target: 28; IgG: 38)

If I do a standard curve, I will probably see this:

Control (Input: 500ug; Target: 2ug; IgG: 0.001ug)
Treatment (Input: 500ug; Target: 8ug; IgG: 0.004ug)

So there's definitely enrichment,
as Control Target / Input = 2ug/500ug=0.4%
vs Treatment Target / Input = 8ug/500ug=1.6%

And comparing the IgG, Ct of 38 vs 40 seems like a 4-fold difference, but when converted to absolute values, 0.001ug and 0.004ug (compared with 2ug and 8ug) are both negligable....

Of course, I made up all these numbers, but conceptually, I think now I understand, if the Ct value of IgG is much larger than your target, there's no reason to worry about~

-edu-

QUOTE (edu @ Dec 19 2008, 01:58 PM)
Hi, i am new in doing MeDIP/ CHIP followed by Realtime PCR .
I saw u did same stuffs. Would u mind to share the protocol and the problems you faced regarding specificity of CHIP, and did u make std curve? if yes, then is it with serial dilutions of DNA from another cells?

thanks.

Now I'm kind of okay with specificity, my IgG control usually has a CT much bigger than my target

When I do standard curve, I use some sonicated DNA samples from the same cells that I collected in the past when I was still optimizing the sonication condition

Of course you need to process the sonicated DNA before using as a standard (reverse crosslink at 65C 5M NaCl overnight, 1hr protease K, then phenol/chloroform, ethanol precipitation, then finally resuspend with H2O)

then I do 10 fold serial dilution, and make sure the standard curve has a slope of about -3.33

if the slope is more negative, it could be due to low efficiency of the primers, if less negative, check if there's primer dimers

besides, my starting concentration of standard curve is 100ng, and usually has a CT of about 20

-jiro_killua-