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Chip-qPCR calculation method

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#1 yg_eagle



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Posted 25 June 2009 - 11:38 AM

Hi, all, I read some paper about how to do Chip-qPCR calculation as follows. But I can not understand at all. Is it correct and could someone explain it for me? Thanks a lot in advance!

Input and immunoprecipitated DNA amplification reactions were run in triplicate in the presence of SYBR-Green (Applied Biosystems). Ct values from each sample were obtained using the Sequence Detector 1.1 software. Relative quantification of template was performed as described previously by Chakrabarti et al. (2002Posted Image) and by the Applied Biosystems manual, with some modifications. Briefly, a Posted ImageCt value representing the difference between control Ct and experimental Ct (acute or chronic) was calculated, using the formula: Posted ImageCt = (Nacute,chronic - Navecontrol) x Ctavecontrol, where N is the normalized Ct value of H4 [Ct(H4)/Ct(Input)] or of H3 [Ct(H3)/Ct(Input)], Nave is the mean N value for the control, and Ctave is the mean Ct value for the control. Fold differences (acute or chronic ChIP relative to control ChIP) were then determined by raising 2 to the Posted ImageCt power. Mean and SEM values were determined for each fold difference, and these values were used in two-tailed paired t tests (which were adjusted for multiple comparisons) to determine statistical significance (p < 0.05). Each PCR reaction, run in triplicate for each brain sample, was repeated at least two independent times.

The Journal of Neuroscience, June 16, 2004, 24(24):5603-5610
Histone Modifications at Gene Promoter Regions in Rat Hippocampus after Acute and Chronic Electroconvulsive Seizures
Nadia M. Tsankova, Arvind Kumar, and Eric J. Nestler

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