NFkB Signaling Pathway ChIP PCR Array

NFκB Signaling Pathway ChIP PCR Array The Mouse NFκB Signaling Pathway EpiTect ChIP qPCR Array profiles the histone modification status or “histone code” of 84 genes related to NFκB-mediated signal transduction. Histone modifications regulate chromatin structure and correlate with the transcriptional activity of associated genes. The NFκB family is a group of transcription factors activated by stress and inflammatory responses. Other NFκB stimuli include growth factors, cytokines, pathogen-associated molecular products (PAMPs), and DNA damage. NFκB-responsive genes regulate processes such as apoptosis, tissue remodeling, tumorigenesis, and inflammation. The NFkB transcriptional response also involves epigenetic factors such as histone deacetylases (HDACs) to enhance NFκB action on target gene promoters. Analyzing histone modifications of the NFκB pathway and NFκB target genes simultaneously can provide insights into the molecular and epigenetic mechanisms regulating NFκB signaling. This array includes members of the Rel, NFκB, and IκB families, NFκB targets, and the extracellular ligands and cell surface receptors that activate this pathway. Using chromatin immunoprecipitation and this real-time PCR array, research studies can easily and reliably analyze the histone modification patterns associated with a focused gene panel involved in NFκB signal transduction.

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Activation of the NFκB Pathway: Ligands and Receptors: Card10, Myd88, Tlr1, Tlr2, Tlr3, Tlr4, Tlr6, Tnf, Tnfsf14. Kinases: Chuk (IKKa), Irak1. IκB kinase and the NFκB Cascade:  Bcl10, Ikbkb, Ikbke, Ikbkg, Myd88, Tlr4. Inflammatory Response:  Nfkb1, Myd88, Tlr1, Tlr2, Tlr3, Tlr4, Tlr6. Positive Regulation of IκB Kinase and the NFκB Cascade: Ligands and Receptors: Cd40 (Tnfrsf5), F2r, Fasl (Tnfsf6), Ltbr, Slc20a1, Tnfrsf1a, Tnfsf10 (Trail). Membrane Molecules: Gja1, Lpar1 (Edg2). Kinases: Ikbke, Ripk1, Tbk1. Other: Bcl10, Bcl3, Nfkbia (IkBa/Mad3), Nlrp12 (Nalp12), Nod1 (Card4), Casp1 (Ice), Casp8 (Flice), Cflar (Casper), Fadd, Malt1, Rel, Tnfaip3, Tradd, Traf3. NFkB Responsive Genes: Cytokines: Ccl2 (Mcp-1), Ccl5 (Rantes), Csf1 (MCSF), Csf2 (GM-CSF), Csf3 (GCSF), Ifng, Irf1, Lta (Tnfb), Tnf. Apoptosis: Bcl2a1a (Bfl-1/A1), Bcl2l1 (Bcl-x), Birc3 (c-IAP3). Adhesion: Icam1. Transcription Factors: Ifng, Irf1. Other Factors Involved in the NFκB Pathway: Ligands and Receptors: Ccl2 (Mcp-1), Cd27 (Tnfrsf7), Csf2 (GM-CSF), Csf3 (GCSF), Edg2, F2r, Fasl (Tnfsf6), Htr2b, Ifng, Il10, Il1a, Il1b, Il1r1, Irak2, Lta, Ltbr, Eif2ak2 (Prkr), Raf1, Slc20a1, Tgfbr1, Tlr7, Tlr8, Tlr9, Tnfrsf10b (DR5), Tnfrsf1a, Tnfrsf1b, Cd40 (Tnfrsf5), Tnfsf10, Tollip, Traf2. Other Extracellular and Membrane Molecules: Gja1, Icam1, Smad3. Kinases: Akt1, Irak2, Mapk3, Eif2ak2 (Prkr), Raf1, Ripk1, Ripk2, Tbk1, Tgfbr1, Zap70. Transcription Factors: Atf1, Atf2, Crebbp, Egr1, Elk1, Fos, Ifng, Irf1, Jun, Nfkb2, Rel, Rela, Relb, Smad3, Stat1. Inflammatory Response: Ccl2 (Mcp-1), Il10, Il1a, Il1b, Tlr7, Tlr8, Tlr9, Tnfrsf1b, Tollip. Gene Table

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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. The ChIP PCR Arrays can be used for research on stem cells, cancer, immunology, stem cells, toxicology, biomarker discovery and validation. How it Works Performance Data Application Examples How it Works The ChIP PCR array is a set of optimized real-time PCR primer assays on 96-well or 384-well plates for pathway or disease focused analysis of in vivo protein-DNA interactions. The ChIP PCR array performs ChIP DNA analysis with real-time PCR sensitivity and the multi-genomic loci profiling capability of a ChIP-on-chip. Simply mix your ChIP DNA samples with the appropriate ready-to-use PCR master mix, aliquot equal volumes to each well of the same plate, and then run the real-time PCR cycling program. (Download user manual) What ChIP PCR Array Offers? Function or Disease Focused: Arrays represent a panel of genomic regions relevant to a biological function or disease state. Reliable & Sensitive: Arrays can analyze multiple genomic regions simultaneously with Real-Time PCR precision and sensitivity. Easy to Use Data Analysis: Download an easy-to-use Excel-based data analysis template [here]. Data analysis is based on the ΔΔCt method with normalization of the specific antibody and control IgG raw data to input raw data. Layout and Controls: The PCR Arrays are available in both 96- and 384-well plates and are used to monitor the expression of 84 genes related to a disease state or pathway plus five housekeeping genes. Controls are also included on each array for ChIP DNA quality controls and general PCR performance. You can easily perform a ChIP PCR Array experiment in your own laboratory, or send your samples to us and take advantage of our PCR Array Services. Performance EpiTect Chip qPCR Arrays provide the high sensitivity, specificity and reproducibility using SYBR-based real-time PCR technology. Sensitivity Together with our easy and fast One-Day ChIP kit, ChIP-Grade Antibody Kits, one million cells per assay as starting material provides 100% effective call rates. 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 ChIP Assay using the EpiTect Chip One-Day Kit and anti-H3K4me3 Antibody Kit. One million cells were used as starting material for each ChIP Assay. 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. Reproducibility The complete ChIP PCR Array System demonstrates a high degree of reproducibility across technical replicates, lots, instruments, and different handling, insuring reliable detection of differences in genomic DNA enrichment among biological samples. Figure 5. 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 EpiTect Chip One-Day Kit. The obtained ChIP DNA samples were characterized in triplicates with EpiTect Chip qPCR primers 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 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 6. Consistent Performance with Various Amount of DNA Samples, Instruments or Handling Conditions. All experiments were performed in triplicates. Cells from MCF-7 (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 96-well plate or 384-well plate format, on a Stratagene MX 3005 or an ABI 7900 Real-Time PCR instrument respectively. The same ChIP DNA samples were used which were stored for extended periods of time as indicated. The results demonstrate high reproducibility of PCR performance across technical replicates, lots, instruments, and differential handling. Specific and Accurate ChIP-qPCR Detection One prerequisite for ChIP PCR Array technology is the uniform and high PCR amplification efficiency allowing a reciprocal comparison of 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 EpiTect Chip qPCR Arrays. A: B: Figure 7. Uniform Amplification Efficiency and Specific PCR Detection. 96 ChIP-qPCR primers were randomly picked from our 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 uniform high amplification efficiency ensures accurate analysis of multiple genomic loci simultaneously using 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 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 PCR product was further validated on agarose gel for a single product of the predicted size without secondary products such as primer dimers Application Examples EpiTect Chip qPCR Arrays provide streamlined approaches to 1) Study biology or disease-focused gene regulation through histone modification and transcriptional regulatory network; 2) Monitor the dynamics of chromatin structure in the screening of function-specific epigenetic patterns; 3) Validate ChIP-on-chip or ChIP-seq results. The EpiTect Chip qPCR Arrays are also powerful tools for studying the mechanism contributing to gene expression changes observed by RT² Profiler PCR Arrays. Below are listed a few examples of application data generated by our R&D group. To see the research using ChIP PCR Arrays published by the scientific community, please see our Publication List: http://www.sabiosciences.com/support_publication.php Stem Cell Research Stem cell differentiation into specific tissues involves the complex yet coordinated action of many transcription factors regulating not only tissue-specific genes, but also genes essential for differentiation itself. Histone modifications at the promoters of transcription factors are key mechanism regulating their expression. We used EpiTect Chip qPCR Arrays and RT² PCR Arrays 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). This RA treatment differentiates pluripotent P19 cells into somatic cells (Figure 2). The EpiTect Chip qPCR Array data showed that both gene expression and histone modifications on key transcription factors were changed in a dynamic manner through the course of P19 cell differentiation (Figure 3). Figure 1. Schematic Representation of Pluripotency-Associated Gene Dynamics throughout Stem Cell 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. ChIP PCR Arrays and RT² PCR Arrays were used to monitor the changes in gene expression levels and histone modification marks (H3Ac, H3K4me3, H3K27me3, and H3K9me3). 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 were preloaded on the ChIP PCR Array. Cluster analysis (http://www.sabiosciences.com/chippcrarray_data_analysis.php) of histone marks and mRNA level changes for the 84 genes were visualized as a heat map to represent the fold-differences during the RA-induced differentiation at the specified time points. Characterize the Pattern of Histone Modifications EpiTect Chip qPCR Arrays can be used to monitoring differential histone modifications across a gene. Figure 4. The Custom EpiTect Chip 30Kb Tiling Array Quickly Maps Histone Modifications Surrounding the Transcription Start Site (TSS) of CDKN1A Gene. EpiTect Chip Antibodies against modified histones (H3Ac, H3K4me2, H3K27me3), or NIS were used to precipitate chromatin from one million HeLa cells. Each ChIP DNA fraction was analyzed with Custom EpiTect Chip 30Kb Tiling Array representing 30 one-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 marks for transcriptional inactive genes (H3K27me3) in the genomic region surrounding the TSS of CDNK1A. Back to Top

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User Manual EpiTect Chip PCR Array User Manual (PDF) Data Analysis Free ChIP PCR Array Data Analysis Software (Excel based) Other Needed Materials Accessories, Equipment & Reagents Required for ChIP PCR Array Web Seminars Attend a live on-line seminar on EpiTect Chip qPCR System Back to Top

Functional Gene Grouping

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Reagents & Software

The complete EpiTect Chip qPCR Array System insures that you will get high-quality results from your ChIP analysis the first time, and every time guaranteed. The Complete EpiTect Chip qPCR Array System Includes: EpiTect Chip One-Day Kit: (GA-101) Sufficient for 12 Chromatin Immunoprecipitations & DNA Purifications EpiTect Chip Antibody Kit Experimentally validated ChIP-grade antibody of interest, control IgG, and control PCR primer sets are included. EpiTect Chip qPCR Array Simultaneous analysis of multiple genomic sites bound to proteins with Real-Time PCR precision. RT² SYBR Green qPCR Master Mix Match with your instrument, 200-reaction scale Order one mix for each pair of 96-well or each individual 384-well PCR Array ChIP PCR Data Analysis - Free Back to Top