# CHIP calculation. confused! - (Nov/04/2007 )

what I know now is I have CT.input CT.target gene, and CT.negative control (IgG)

then I calculate as following
dCT.target gene = CT.i - CT.taget gene
dCT.IgG = CT.i - CT.IgG

dSD.tr = sqrt( (SD.i)^2 + (SD.tr)^2 ) / sqrt(n)
dSD.nc = sqrt( (SD.i)^2 + (SD.nc)^2 ) / sqrt(n)
where n=number of replicate qPCR wells per sample (usually n=3)

Then I have

ddCT = dCT.targent gene - dCT.IgG
ddSD = sqrt( (dSD.tr)^2 + (dSD.nc)^2 ).

Then I am confused!!!! about fold enrichment. some People calculate fold enrichment with each antibody relative to either no Antibody, or some one said relative to PI sample (what is PI sample?) or some people calculate enrichment relative to genes this antibody should not bind (iE. GAPDH, or whatever you knows not bind at all)

Which one is right????????? I am so so confused.

-cathyli-

QUOTE (cathyli @ Nov 4 2007, 07:34 AM)

what I know now is I have CT.input CT.target gene, and CT.negative control (IgG)

then I calculate as following
dCT.target gene = CT.i - CT.taget gene
dCT.IgG = CT.i - CT.IgG

dSD.tr = sqrt( (SD.i)^2 + (SD.tr)^2 ) / sqrt(n)
dSD.nc = sqrt( (SD.i)^2 + (SD.nc)^2 ) / sqrt(n)
where n=number of replicate qPCR wells per sample (usually n=3)

Then I have

ddCT = dCT.targent gene - dCT.IgG
ddSD = sqrt( (dSD.tr)^2 + (dSD.nc)^2 ).

Then I am confused!!!! about fold enrichment. some People calculate fold enrichment with each antibody relative to either no Antibody, or some one said relative to PI sample (what is PI sample?) or some people calculate enrichment relative to genes this antibody should not bind (iE. GAPDH, or whatever you knows not bind at all)

Which one is right????????? I am so so confused.

-cathy-

Hi,

I can tell you how I calculate my ChIP-PCR. Maybe that helps you (we use LightCycler).

So, I have the target gene X and the anitibody against the protein Y which binds to X. I have different treatments for cells. One treatment/no treatment is a kind of negative control where Y should not bind X (an acceptabel background for X-bound Y). And I have "no-antibody" control. I dilute one of the inputs (strong signal) 1:10exp1, 1:10exp2, 1:10exp3 and 1:10exp4 (if necessary). The standard curve will be CP values of these against 1, 2, 3, 4. This gives a falling linear curve with the formula y (CP)=-ax (log concentration)+b. According to that I calculate log concentrations aof samples and inputs. From log conc. I calculate concentrations and normalize to the inputs (sample/input). The treatment I mentioned above (where Y should not bind X) is displayed as "1" and all the other normalzed values are shown as fold change. The negative control (no-ab) is just my intern control. I don`t take it into account in the calculation.
I send you a PM to make it more clear.

Hope that helps.

-zek-