EpiTect ChIP antibodies against modified histones (H3Ac, H3K4me2, H3K27me3), or Control IgG (NIS; non-immune serum) were used to precipitate chromatin from 1 million HeLa cells. Each ChIP DNA fraction was analyzed with an EpiTect ChIP Custom qPCR Array representing thirty 1-kb tile intervals across the promoter region of the CDKN1A gene. The results indicate the enrichment of histone markers for actively transcribed genes (H3Ac and H3K4me2), but not histone markers for transcriptionally inactive genes (H3K27me3) in the genomic region surrounding the transcription start site (TSS) of CDKN1A.
Sonicated chromatin from HeLa cells (20 µg) was immunoprecipitated with 2 µg anti-H3ac antibody or control IgG for 2 hours using the EpiTect ChIP OneDay Kit. The obtained ChIP DNA samples were characterized in triplicate with EpiTect ChIP qPCR Assays specific for the active genes (GAPDH, RPL30, ALDOA), inactive genes (MYOD1, SERPINA), repetitive sequence (SAT2, SATa), and an ORF-free region (IGX1A) either within the same array plate or among different array plates to evaluate the intra- and inter-plate consistency. The anti-H3ac antibody enriched genomic DNA at active gene promoter regions with a high signal-to-noise ratio and a low coefficiency of variation (less than 2.02%), irrespective of the type of assay (intra- or inter-plate).
All experiments were performed in triplicates. Chromatin from MCF-7 cells (1 million per sample) were subjected to ChIP assay with anti-RNA Polymerase II (Pol 2) antibody followed by real-time PCR analysis of the proximal promoter of GAPDH, and an ORF-free region (IGX1A). Users A and B performed the PCR assays either in 96-well or 384-well plate format, on a Stratagene Mx3005 or an ABI 7900 real-time PCR instrument, respectively. The same ChIP DNA samples were used after storage for extended periods of time as indicated. The results demonstrate high reproducibility of PCR performance across technical replicates, lots, instruments, and differential handling.
A panel of 96 EpiTect ChIP qPCR Assays were randomly selected from the genomewide primer pool and analyzed for performance. Upper panel: All assays exhibit an average amplification efficiency of 99% with a 104.5% CI between 102.5–105.2%. The uniformly high amplification efficiency ensures accurate analysis of multiple genomic loci simultaneously using the ΔCT method. Lower panel: Each EpiTect ChIP qPCR Assay is experimentally verified using dissociation (melt) curve analysis and agarose gel analysis. Each EpiTect ChIP qPCR Assay on an EpiTect ChIP qPCR Array produces a single specific product as indicated by a single dissociation curve peak at a melting temperature (Tm) greater than 75ºC, and is further verified by agarose gel for a single product of predicted size.