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EpiTect ChIP qPCR Arrays

For application-focused quantitative detection of gene panels in chromatin immunoprecipitation experiments

  • Simultaneous analysis of 84 pathway-focused genes
  • Requires as few as 1 million cells per array
  • Streamlined procedure, ready to use on most cyclers
EpiTect ChIP qPCR Arrays enable the simultaneous analysis of DNA–protein interactions using chromatin immunoprecipitation (ChIP)-enriched genomic DNA across a focused panel of genes. The carefully selected gene panels included in the EpiTect ChIP qPCR Arrays represent an epigenetically regulated biological pathway or disease state.
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EpiTect ChIP 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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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.

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 verification of every EpiTect ChIP qPCR Assay, and high performance RT2 SYBR® Green Mastermix ensures the superior performance of EpiTect ChIP qPCR Arrays. See figure "Uniform amplification efficiency and specific PCR detection".

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.

EpiTect ChIP qPCR Arrays represent a focused panel of genomic regions relevant to a biological function or disease state. The target-sequence-specific assays in each well are designed with a proprietary algorithm developed to specifically to address the challenges presented by genomic DNA sequences. Each assay is also experimentally verified for the high amplification efficiency and specificity expected of optimal SYBR Green-based real-time PCR assays. The performance of the EpiTect ChIP qPCR Arrays is guaranteed when used with the appropriate RT2 SYBR Green qPCR Mastermix.

EpiTect ChIP PCR Arrays are available in both 96- and 384-well plates and are used to monitor the binding of a target protein to the genomic loci of 84 different genes related to a disease state or pathway. Positive and negative controls for protein-specific immunoprecipitation are also included on each array, as well as controls for general PCR performance.

Available array formats

EpiTect ChIP qPCR Arrays are available in the following formats:

EpiTect ChIP qPCR Array Format A: 96-well plates containing dried qPCR primer assays, Optical Thin-Wall 8-Cap Strips
EpiTect ChIP qPCR Array Format C: 96-well plates containing dried qPCR primer assays, Optical Adhesive Film
EpiTect ChIP qPCR Array Format D: 96-well plates containing dried qPCR primer assays, Optical Thin-Wall 8-Cap Strips
EpiTect ChIP qPCR Array Format E: 384-well plate(s) containing dried qPCR primer assays, Optical Adhesive Film, 384EZLoad Covers
EpiTect ChIP qPCR Array Format F: 96-well plates containing dried qPCR primer assays, Optical Adhesive Film
EpiTect ChIP qPCR Array Format G: 384-well plate(s) containing dried qPCR primer assays, Optical Adhesive Film, 384EZLoad Covers
Procedure

Simply separately mix the input DNA fraction and specific antibody and control IgG ChIP DNA fractions with the appropriate ready-to-use RT2 SYBR Green qPCR Mastermix, aliquot equal volumes of each fraction into each well of its 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 ChIP DNA fraction to the input DNA fraction raw data. An easy-to-use, Excel-based data analysis template for these calculations  makes analysis simple.

Applications

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

EpiTect ChIP 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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Epithelial to Mesenchymal Transition (EMT) EpiTect ChIP qPCR Array
Introduction
The Human Epithelial to Mesenchymal Transition (EMT) EpiTect Chip qPCR Array profiles the histone modification status or “histone code” of 84 genes key to Epithelial to Mesenchymal Transition (EMT). Histone modifications define chromatin structure, thus playing a key role in the epigenetic-based regulation of gene expression. EMT and the reciprocal mesenchymal to epithelial transition (MET), both mediated by gene expression changes, are key processes involved in both tumor metastasis and stem cell differentiation and development. During EMT, epithelial cells lose apical/basolateral polarity, break intercellular tight junctions, and degrade basement membrane extracellular matrix components to become migratory mesenchymal cells. As such, the array represents cell surface receptor, extracellular matrix, and cytoskeletal genes mediating cell adhesion, migration, motility, and morphogenesis; genes controlling cell differentiation, development, growth, and proliferation; as well as signal transduction and transcription factor genes that cause EMT and all of its associated processes. The results can help you provide further insights into the role of histone modifications in regulating EMT-dependent tumor metastasis and stem cell differentiation and development. Using chromatin immunoprecipitation and this real-time PCR Array, your research study can easily and reliably analyze the histone modification patterns associated with a focused gene panel involved in EMT.
Gene List

Genes Up-Regulated During EMT: AHNAK, BMP1, CALD1, CDH2 (N-cadherin), COL1A2, COL3A1, COL5A2, FN1, FOXC2, GNG11, GSC, IGFBP4, ITGA5, ITGAV, MMP2 (Gelatinase A), MMP3, MMP9 (Gelatinase B), MSN, SERPINE1 (PAI-1), SNAI1, SNAI2, SNAI3, SOX10, SPARC, STEAP1, TCF4, TIMP1, TMEFF1, TMEM132A, TWIST1, VCAN, VIM, VPS13A, WNT5A, WNT5B.

Genes Down-Regulated During EMT: CAV2, CDH1 (E-cadherin), DSP, FGFBP1, IL1RN, KRT19, MITF, MST1R, NUDT13, PPPDE2, RGS2, SPP1 (Osteopontin), TFPI2, TSPAN13.

Genes with Known Histone Modifications during EMT:
Increased H3K4me3: AHNAK, AKT1, BMP1, CALD1, CAV2, CDH2 (N-cadherin), CTNNB1, FN1, FZD7, GNG11, GSK3B, IGFBP4, ILK, ITGA5, MAP1B, MITF, MMP2 (Gelatinase A), RGS2, SERPINE1 (PAI-1), SNAI1, SNAI2, SPARC, TCF4, TGFB1, TGFB2, TGFB3, TIMP1, TMEFF1, TSPAN13, VIM, VPS13A, WNT5A.
Decreased H3K27me3: DSP, FGFBP1, GSC, IL1RN.

Differentiation & Development: AKT1, BMP1, BMP2, BMP7, COL3A1, COL5A2, CTNNB1, DSP, ERBB3, F11R, FOXC2, FZD7, GSC, KRT14, MITF, MST1R, NODAL, NOTCH1, PTP4A1, SMAD2, SNAI1, SNAI2, SOX10, TGFB2, TGFB3, TMEFF1, TWIST1, VCAN, WNT11, WNT5A, WNT5B.

Morphogenesis: CTNNB1, FOXC2, PPP3R1, RAC1, SMAD2, SNAI1, SOX10, TGFB1, TGFB2, TGFB3, TWIST1, WNT11, WNT5A.

Cell Growth & Proliferation: AKT1, BMP1, BMP2, BMP7, CAV2, CTNNB1, EGFR, ERBB3, FGFBP1, FOXC2, HIF1A, IGFBP4, ILK, MST1R, NODAL, PDGFRB, TGFB1, TGFB2, TGFB3, TIMP1, VCAN.

Migration & Motility: CALD1, CAV2, EGFR, FN1, ITGB1, MSN, MST1R, NODAL, PDGFRB, RAC1, STAT3, TGFB1, VIM.

Cytoskeleton: CAV2, KRT7, MAP1B, PLEK2, RAC1, VIM.

Extracellular Matrix & Cell Adhesion: BMP1, BMP2, BMP7, CDH1 (E-cadherin), CDH2 (N-cadherin), COL1A2, COL3A1, COL5A2, CTGF, CTNNB1, DSC2, EGFR, ERBB3, F11R, FN1, FOXC2, ILK, ITGA5, ITGAV, ITGB1, MMP2 (Gelatinase A), MMP3, MMP9 (Gelatinase B), PTK2, RAC1, SERPINE1 (PAI-1), SPP1 (Osteopontin), TGFB1, TGFB2, TIMP1, VCAN.

Signaling Pathways:
Estrogen Receptor: CAV2, ESR1 (ERa), KRT19, TGFB3.
G-Protein Coupled Receptor: AKT1, FZD7, GNG11, RAC1, RGS2.
Integrin-Mediated: COL3A1, CTGF, ILK, ITGA5, ITGAV, ITGB1, PTK2.
Notch: FOXC2, NOTCH1.
Receptor Tyrosine Kinase: EGFR, ERBB3, PDGFRB, RGS2, SPARC.
TGFb / BMP: BMP1, BMP2, BMP7, COL3A1, SMAD2, SMAD4, TGFB1, TGFB2, TGFB3.
WNT: CTNNB1, FZD7, GSK3B, WNT11, WNT5A, WNT5B.

Transcription Factors: CTNNB1, ESR1 (ERa), FOXC2, GSC, MITF, NOTCH1, SIP1, SMAD2, SNAI2, SNAI3, SOX10, STAT3, TCF4, TWIST1, ZEB2.

Gene Resource List
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Name Human Epithelial to Mesenchymal Transition (EMT) EpiTect ChIP qPCR Array (GH-090A)