• Specificity: Experimentally-validated genome wide primer assays
• Reliability: Consistent, reproducible results
• Sensitivity: As little as 1 million cells per ChIP array (25 to 50 µg chromatin)

Specific and Accurate ChIP-qPCR Detection
ChIP PCR Array technology relies on uniformly high PCR amplification efficiencies to accurately compare ChIP enrichment among all genomic loci analyzed. The unique combination of SABiosciences' proprietary ChIP-qPCR primer design algorithm, rigorous validation of every ChIP-qPCR primer assay, and high performance SYBR Green master mix guarantees superior performance of ChampionChIP PCR Arrays.

A:

B:

Figure 1. Uniform Amplification Efficiency and Specific PCR Detection. A panel of 96 ChIP-qPCR primers were randomly selected from the genome-wide primer pool and analyzed for their performance. (A) All assays exhibit an average amplification efficiency of 99% with a 104.5% confidence interval between 102.5-105.2%. The uniformly high amplification efficiency ensures accurate analysis of multiple genomic loci simultaneously using the ΔΔCt method. (B) Each ChIP-qPCR primer assay is experimentally validated using dissociation (melt) curve analysis and agarose gel verification. Each pair of primers on ChampionChIP PCR Array produces a single specific product as indicated by a single dissociation curve peak at a melting temperature (Tm) greater than 75 ºC, and is further validated by agarose gel for single product of predicted size.

Reproducibility
The complete ChampionChIP PCR Array System demonstrates a high degree of reproducibility across technical replicates, production lots, real-time PCR instruments, and different end-users, ensuring reliable detection of differences in genomic DNA enrichment among biological samples.

Figure 2. Consistent Performance within the Same Plate or across Different Plates. Sonicated chromatin from HeLa cells (20 µg) was immunoprecipitated with 2 µg of anti-H3ac antibody or control IgG for 2 hours using the ChampionChIP One-Day Kit. ChIP DNA samples were characterized in triplicates with ChampionChIP qPCR primers specific for 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 in order 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 co-efficiency of variation (less than 2.02%), irrespective of the type of assay (intra or inter-plate).

Figure 3. Consistent Performance with Various Amount of DNA Samples, Instruments or Handling Conditions. All experiments were performed in triplicates. MCF-7 cells (1 million per sample) were subjected to ChIP assay with anti-RNA Polymerase II (Pol 2) antibody followed by qPCR analysis of the proximal promoter of GAPDH, and an ORF-free region (IGX1A). Researcher A & B performed the PCR assays either in a 96-well plate or 384-well plate format on a Stratagene MX 3005 or an ABI 7900 Real-Time PCR instrument respectively, using the same ChIP DNA sample stored as indicated. The results demonstrate high reproducibility of PCR performance.

Sensitivity
Together with the straightforward One-Day ChIP kit and ChIP-Grade Antibody Kits, 100% effective call rates are obtained with ChIP DNA from as little as one million cells per assay.

Ct Range	Percent Distribution of Ct Values
	Input	H3K4me3	Control IgG
<24	0%	27%	0%
25-30	100%	60%	0%
30-35	0%	13%	96%
Absent Calls	0%	0%	4%

Table 1. ChIP PCR Arrays Analyze the Enrichment of 84 Genomic Sites with as Little as One Million Cells. P19 mouse embryonic carcinoma cells were prepared for the ChampionChIP PCR Array using the ChampionChIP One-Day Kit and anti-H3K4me3 Antibody Kit. One million cells were used as starting material for each ChIP Array (ChIP fraction). The purified ChIP DNA samples were characterized using Mouse Stem Cell Transcription Factor ChIP PCR Array with 1/100th of the ChIP DNA as template in each well. The Real-Time PCR results demonstrate 100 % effective call rates for the Input Fraction (Ct < 30). The difference of Ct value between the anti-H3K4me3 antibody and the control IgG fractions indicates the specific enrichment of the antibody, whereas the high Ct value of the control IgG fraction indicates the low background of the assay.

Stem Cell Research

Tissue-specific differentiation of stem cells involves the complex and coordinated actions of many transcription factors regulating genes essential for differentiation and tissue-specific expression. Histone modifications at transcription factor promoters are a key mechanism regulating their expression. ChampionChIP PCR Arrays and RT² Profiler PCR Arrays were used to monitor the dynamic coordination of epigenetic modification and gene expression during retinoic acid (RA) induced differentiation of P19 mouse embryonic carcinoma cells (Figure 1). The RA treatment differentiates pluripotent P19 cells into somatic cells (Figure 2). The ChampionChIP PCR Array data showed that gene expression and histone modifications on key transcription factors dynamically changed during P19 cell differentiation (Figure 3).

Figure 1. Monitoring Pluripotency-Associated Gene Dynamics During Differentiation

Figure 2. Retinoic Acid (RA) Differentiation of Mouse Embryonic Carcinoma P19 Cells.

Figure 3. Dynamic Epigenetic Alternations and Gene Expression Changes during RA-Induced P19 Differentiation. Champion ChIP PCR Arrays and RT² Profiler PCR Arrays were used to monitor gene expression level changes and modified histone binding (H3Ac, H3K4me3, H3K27me3, and H3K9me3). Modified histone binding to the promoter region and expression levels of 84 key stem cell transcription factors were simultaneously analyzed during RA-induced neurogenesis of P19 cells at various time points (day 0, 4, and 8). Primer sets for the +1kb region downstream of the transcription start sites of the 84 genes and 12 control regions are contained in the ChampionChIP pPCR Array. Cluster analysis of histone binding and expression changes for the 84 gene panel represents the fold-differences observed during RA-induced differentiation at the specified time points.

ChIP qPCR Data Analysis

SYBR Green Master Mixes

Negative Control PCR Primers

Positive Control PCR Primers

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