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EpiTect ChIP Custom qPCR Arrays

For quantitative detection of custom-defined gene panels in chromatin immunoprecipitation experiments

  • qPCR design for any 96- or 384-well qPCR instrument
  • Rapidly verify ChIP-on-chip or ChIP-Seq data
  • Study multiple transcription factor binding sites

EpiTect ChIP Custom qPCR Arrays enable the simultaneous analysis of DNA–protein interactions using chromatin immunoprecipitation (ChIP)-enriched genomic DNA across a focused panel of genomic loci tailored to specific research interests. Analyze multiple gene-specific binding sites for the same transcription factor, the histone code at the proximal promoter of multiple genes, or the histone code or binding sites for multiple transcription factors across one entire gene promoter.

Cat No./ID: 334111
EpiTect ChIP Custom qPCR Array
96-well plate or 384-well plate containing assays, film or cap strips

EpiTect ChIP Custom qPCR Arrays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Monitoring of differential histone modifications.

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.

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Consistent intra- and inter-plate performance.

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).

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Consistent performance with different amounts of DNA, instruments, or handling conditions.

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.

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Uniform amplification efficiency and specific PCR detection.

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.

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EpiTect ChIP Custom qPCR Array formats.
Performance

EpiTect ChIP qPCR Arrays provide high sensitivity, specificity, and reproducibility using SYBR® Green-based real-time PCR technology.

Reproducibility

The complete EpiTect ChIP qPCR Array system demonstrates a high degree of reproducibility across technical replicates, lots, and instruments. This consistency ensures reliable detection of differences in genomic DNA enrichment among biological samples. See figures "Consistent intra- and inter-plate performance" and "Consistent performance with different amounts of DNA, instruments, or handling conditions".

Specific and accurate ChIP-qPCR detection

One prerequisite for ChIP qPCR array technology is uniform and high PCR amplification efficiency, which allows a reciprocal comparison of ChIP enrichment among all genomic loci analyzed. The unique combination of a proprietary ChIP-PCR primer design algorithm, rigorous experimental validation of every EpiTect ChIP qPCR Assay, and high performance SYBR® Green Mastermix ensures the superior performance of EpiTect ChIP qPCR Arrays. See figure "Uniform amplification efficiency and specific PCR detection".

Analyzing the histone code across an entire gene promoter

One application of EpiTect ChIP Custom qPCR Arrays is the monitoring of differential histone modifications across a gene. The EpiTect ChIP Custom qPCR Array quickly maps histone modifications surrounding the transcription start site (TSS) of the CDKN1A gene (see figure "Monitoring of differential histone modifications").

Principle

Regulating gene expression requires dynamic but regulated interactions between genomic DNA and proteins, such as transcription factors, coactivators, corepressors and modified histones. One important technique for studying the discovery of new and the timing and extent of known in vivo protein-DNA binding is chromatin immunoprecipitation (ChIP). Chemical crosslinking at the end of a biological experiment covalently captures and freezes the protein–DNA interactions. Sonication shears the chromatin into manageable sizes for the sensitive detection of specific genomic DNA sequences. Standard immunoprecipitation pulls down a target protein of interest and its bound DNA. Crosslink reversal and DNA purification, followed by real-time PCR detection of specific sequences then quantifies the amount of protein-bound DNA.

The EpiTect ChIP Custom qPCR Array contains a set of optimized real-time PCR primer assays on 96-well or 384-well plates for analysis of in vivo protein–DNA interactions on custom-defined gene content tailored to a specific research interest. The EpiTect ChIP qPCR Assays used in the EpiTect ChIP Custom qPCR Arrays are genomewide, laboratory-verified, real-time PCR tools to analyze any promoter region in human, mouse, and rat samples. The EpiTect ChIP Custom qPCR Array performs ChIP DNA analysis with real-time PCR sensitivity and the multi-genomic loci profiling capability of a ChIP-on-chip. It allows the simultaneous analysis of multiple genomic loci and samples in 96- or 384- well format on any real-time PCR instrument. The performance of the EpiTect ChIP Custom qPCR Arrays is guaranteed when used with the appropriate RT2 SYBR Green qPCR Mastermix.

Fully customizable arrays

EpiTect ChIP Custom qPCR Arrays are available in a series of 96- and 384-well formats (see figure "EpiTect ChIP Custom qPCR Array formats"). Different formats are also possible. Please inquire for more information and to place an order. EpiTect ChIP Custom qPCR Arrays are manufactured, laboratory-verified, and shipped in 2–3 weeks from order verification.

Procedure

Separately mix input DNA fractions and specific antibody and control IgG ChIP DNA samples with the appropriate ready-to-use PCR Mastermix, aliquot equal volumes to the correct and designated wells of the same plate or into each well of a fraction’s own plate, and then run the recommended real-time PCR cycling program.

 

Data analysis is based on the ∆CT method to calculate percent enrichment with normalization of the specific antibody and control IgG ChIP DNA fraction to the input DNA fraction. An easy-to-use Excel-based data analysis template for these calculations can be tailor-made.

Applications

EpiTect ChIP qPCR Arrays can be used for research into cancer, immunology, stem cells, toxicology, and biomarker discovery and validation, and are also powerful tools for studying regulatory mechanisms behind the gene expression changes observed with RT² Profiler PCR Arrays and Assays. EpiTect ChIP Custom qPCR Arrays provide a reliable tool for the analysis of a panel of ChIP-enriched genomic sequences associated with transcription factors and coregulators, modified an unmodified histones, and other DNA-binding proteins.

EpiTect ChIP Custom qPCR Arrays also streamline:

Identification of disease biomarkers
Characterization of signal transduction pathways
Examination of transcriptional regulation
Detection of epigenetic changes
Verification of ChIP-on-chip and ChIP-Seq data
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