T Helper Cell Differentiation EpiTect

ChIP qPCR Array

辅助T细胞分化ChIP qPCR芯片
 

Product	Species	Technology	Cat. No.
T Helper Cell Differentiation EpiTect ChIP qPCR Array	Human	Histone Modifications	GH-503A
T Helper Cell Differentiation EpiTect ChIP qPCR Array	Mouse	Histone Modifications	GM-503A

  The Human T Helper Cell Differentiation EpiTect Chip qPCR Array profiles modified histone and nuclear protein binding to the proximal promoter of a panel of genes regulating the commitment of precursor T cells into specific effector subtypes. The array contains 96 pairs of qPCR primers targeting the 1-kb region downstream of the transcription start sites (TSS) of 84 biological important genes plus 12 appropriate control regions. The genes represented by this array include cytokines, cytokine receptors, transcription factors, and other signaling molecules regulating differentiation into Th1 or Th2 cells as well as specific makers for these subtypes. Profiling the histone modifications and nuclear protein binding events at these gene promoters in your T cell populations will help you correlate these interactions with subtype-specific cellular (Th1) and humoral (Th2) immune responses and immune disorders, such as allergy, asthma, autoimmunity, diabetes, hypersensitivity, and rheumatoid arthritis. The results also provide insights into the epigenetic molecular mechanisms and biological pathways behind T helper cell lineage in your model system. Using Chromatin Immunoprecipitation and this real-time PCR Array, you can easily and reliably analyze the association between your chosen nuclear factors and the promoters for a focused gene panel involved in T helper cell differentiation。
  辅助T细胞分化ChIP qPCR芯片用于研究前体T细胞分化成特定功能亚群过程的相关基因近端启动子被修饰组蛋白和核酸蛋白绑定的情况。该芯片包含96对qPCR引物，这些引物针对84个生物学重要基因的转录起始站点(TSS) 下游1 kb区域，以及12个对照域。这个芯片的基因包括细胞因子、细胞因子受体、转录因子和其他调节分化成Th1、Th2细胞的信号分子及这些亚型的分子标记。分析T细胞亚群中组蛋白修饰和核蛋白绑定这些基因启动子，有助于将这些交互作用与亚型特异细胞(Th1)和体液细胞(Th2)免疫反应和过敏、哮喘、自身免疫、糖尿病、过敏和风湿性关节炎等免疫疾病相关联。得到的结果同样有助于对研究者模型中辅助T细胞分化分子机制和生物学通路的深入研究。使用实时定量PCR，可以简单可靠地同时分析你选择的核因子与辅助T细胞分化相关基因启动子的关系。
Cytokines & Receptors:CCL5, CCL7, CCR3, CCR4, CCR5, CCR6, IL12B, IL12RB2, IL13, IL13RA1, IL17A, IL17RE, IL18, IL18R1, IL18RAP, IL1R1, IL1R2, IL1RL1, IL2, IL21, IL2RA, IL4, IL4R, IL5, IL9, TNF.
T Helper 1 Subtype Markers:CCR5, HAVCR2, IGSF6, IL12B, IL18, IRF1, SOCS1, SOCS5, TLR4, TLR6, TNF.
T Helper 2 Subtype Markers:CCL5, CCL7, CCR3, CCR4, CEBPB, GFI1, GPR44, ICOS, IL13RA1, IL4R, JAK1, NFATC1, NFATC2.
Transcription Factors:CEBPB, FOSL1, FOXP3, GATA3, GATA4, HOXA10, HOXA3, ID2, IRF4, IRF8, MAF, NFATC1, NFATC2, NR4A1, NR4A3, POU2F2, REL, RELB, RORA, RORC, RUNX1, RUNX3, STAT1, STAT6, TOX, ZBTB7B.
Genes Differentially Expressed and Enriched with H3K4me3 but not with H3K27me3:
Th1 Cells:EOMES, IFNG, IL12RB2, IL18R1, IL18RAP, FASLG, TBX21.
Th2 Cells:ASB2, GATA3, IL13, IL1RL1, IL4, IL5, PPARG.
Th17 Cells: IL17A, IL17RE, IL1R1, IL21, RORA, RORC.
Inducible and Natural T Regulatory (iTreg and nTreg) Cells:CCL4, CCR6, FOSL1, FOXP3, IKZF2, IL9, IRF4, IRF8, MYB, NR4A1, NR4A3, POU2F2, REL, RELB, TGIF1, TNFSF11.
Genes Differentially Methylated at Their Proximal Promoters in Conventional Versus Regulatory T Cells:CACNA1F, CHD7, FOXP3, GATA4, HOPX, HOXA10, HOXA3, ID2, IKZF2, IL1R2, IL2RA, KIF2C, LRRC32, PERP, PKD2, TNFRSF9, TP53INP1, UTS2.

工作原理：

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.