Chromatin was isolated from 4 million untreated HeLa cells and sonicated. DNA was then purified from an input DNA fraction (diamonds), and immunoprecipitations using either control IgG (triangles), anti-p53 (p53 IP, squares), or RNA Polymerase II (Pol2 IP, circles) using the EpiTect ChIP OneDay Kit. Different volumes of each DNA preparation (1.0, 2.0, and 4.0 µl) were used to obtain CT values and generate a calibration curve. Straight lines indicate that no inhibitors are present in the DNA preparations that interfere with real-time PCR. The results also demonstrate the reliability of the enrichment by the EpiTect ChIP OneDay Kit, since the fold of enrichment is independent of the volumes of template from the immunopreciptated DNA.
Triplicate wells of HCT-116 cells were fixed with formaldehyde. Chromatin was immunoprecipitated using the EpiTect ChIP OneDay Kit. Enriched genomic DNA was then purified and characterized by EpiTect ChIP qPCR Assays for the proximal promoter region of GAPDH and for an ORF-free region as a negative control. The results for each immunoprecipitation are expressed as percentage of input, or the fraction of the total input DNA co-immunoprecipitating with RNA Polymerase II.
Four independent researchers collected duplicate samples of 2 million HCT116 cells. Each performed ChIP with RNA Polymerase II antibody (RNA Pol II) or control IgG from an EpiTect ChIP Antibody Kit. The specific enrichment of ChIP DNA was analyzed using an EpiTect ChIP qPCR Assay for the GAPDH proximal promoter. The results are expressed as percentages of input results (table), their average (chart y-axis), and standard deviation (error bars). Multiple researchers can achieve ChIP-qPCR of Pol II that agrees with only a 4.8% coefficient of variation.