辅助T细胞分化甲基化PCR芯片 T Helper Cell Differentiation EpiTect Methyl qPCR Array

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T Helper Cell Differentiation EpiTect

Methyl qPCR Array

  辅助T细胞分化甲基化PCR芯片

 

Product	Species	Technology	Cat. No.
T Helper Cell Differentiation EpiTect Methyl qPCR Array	Human	DNA Methylation	EAHS-341Z
T Helper Cell Differentiation EpiTect Methyl qPCR Array	Mouse	DNA Methylation	EAMM-341Z
T Helper Cell Differentiation EpiTect Methyl qPCR Array	Rat	DNA Methylation	EARN-341Z

The Human T Helper Cell Differentiation EpiTect Methyl II Signature PCR Array profiles the promoter methylation status of a panel of 22 genes regulating the commitment of precursor T cells to differentiate into specific effector subtypes. The 2 most well-known and characterized T helper cell subtypes, Th1 and Th2 cells, mediate cellular and humoral immune responses, respectively.
Many published research studies find that one subtype population and its immune response tend to dominate in immune disorders such as allergy, asthma, autoimmunity, diabetes, hypersensitivity, and rheumatoid arthritis. This array profiles key regulatory genes encoding mostly transcription factors and cytokines demonstrating differential methylation status or histone modifications during proliferating T cell differentiation into effector Th1 or Th2 cells. Profiling research samples containing genomic DNA from T cell population sources will help to correlate these epigenetic markers with experimentally-induced, subtype-specific immune responses. The results can also provide insights into the epigenetic regulation of the molecular mechanisms and biological pathways behind T helper cell lineage commitment decisions in mammalian model systems. With a simple restriction enzyme digestion and real-time PCR, research studies can analyze the promoter methylation status of 22 different genes involved in T helper cell differentiation with this DNA methylation PCR array
 
辅助T细胞分化甲基化PCR芯片用于研究参与前体T细胞分化成特定功能亚群的22个基因的启动子甲基化状态。Th1细胞和Th2细胞是两个和极具特色的辅助T细胞亚，分别调节细胞和体液免疫反应。许多发表的研究发现其中一个亚群及其免疫反应往往主导免疫疾病如过敏、哮喘、自身免疫、糖尿病、过敏和风湿性关节炎。这个芯片包含编码转录因子和细胞因子的关键调控基因，验证增殖T细胞分化成效应Th1、Th2细胞过程不同的甲基化状态或组蛋白甲基化修饰。分析包含T细胞种群基因组DNA样本将有助于关联这些表观遗传标记与实验性诱导和亚基特异性免疫反应。结果还助于洞察哺乳动物模型系统中辅助T细胞承诺决策的表观遗传调控的分子机制和生物学途径。利用这个芯片，通过简单的限制性内切酶消化和实时定量PCR，就可以研究分析22个不同辅助T细胞分化基因启动子的甲基化状态。
Th1 Cells:EOMES, TBX21.
Th2 Cells:GATA3, IL13, PPARG.
Th17 Cells:RORA.
Inducible & Natural Regulatory T (iTreg & nTreg) Cells:FOSL1, IRF4, IRF8, MYB, NR4A3, POU2F2, REL, RELB, TGIF1, TNFSF11.
Conventional Versus Regulatory T Cells:CHD7, GATA4, HOXA10, ID2, LRRC32, PERP.


工作原理：

How it Works

The Copy Number PCR array is a set of optimized real-time PCR primer assays on 96-well, 100-tube or 384-well plates for measuring changes in copy number. The PCR array performs copy number analysis with real-time PCR sensitivity and the multi-loci profiling capability of a microarray. Simply mix the genomic DNA sample 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)

Figure 1:

How Copy Number PCR Arrays Work - Protocol Chart

 

The procedure involves isolating genomic DNA (QIAGEN QIAamp DNA Mini Kit or FFPE Tissue Kit is recommended), qPCR detection on qBiomarker Copy Number PCR Arrays or Assays, and data analysis (using the qBiomarker Copy Number Data Analysis). An optional DNA sample quality control step immediately before the detection array or assay setup allows the user to qualify the DNA samples.
To complete the Copy Number PCR Array procedure, start with 400 to1000 nanograms of genomic DNA isolated from fresh tissues, or as low as 800 nanograms of DNA from FFPE sections. Then, mix your DNA with the ready-to-use qBiomarker SYBR Green Mastermixes and aliquot the mixture into each well of the same plate containing pre-dispensed locus-specific primer assays. By performing real-time PCR, the copy number status of a particular sample is determined by comparing the CT values between your test sample and a wild-type control sample (see qBiomarker Copy Number Data Analysis section for detailed principles).

Why qBiomarker Copy Number Arrays?
     Guaranteed Performance* - ready-to-use for copy number analysis
     Time and cost saving - less than 30 minutes hands-on time for analyzing 23 loci
     Ease of data analysis using our easy-to-use Excel-based data analysis template or web-based analysis tool

Layout and Controls: The PCR Arrays are available in both 96-, 384-well plates and 100-well discs and are used to measure the copy number of 23 or 95 genes related to a disease state or pathway. Each gene/locus-specific assay is repeated in technical quadruplicate as indicated in the figure above. As an example, the first gene in the array is measured in wells A1, C1, E1 and G1. The qBiomarker Multicopy Reference Assay (MRef) is used to normalize the data for differences in the amounts of genomic DNA. The Multicopy Reference Assay is shown in purple in the above figure in wells B12, D12, F12, and H12.

You can easily perform a Copy Number PCR Array experiment in your own laboratory, or send your samples to us and take advantage of our PCR Array Services.

*:  when using qBiomarker SYBR® Green Mastermix.

Performance Data: 
The qBiomarker Copy Number PCR Array System yields a more accurate representation of changes in copy number by using a multi-copy reference assay to normalize the raw target specific data.

qBiomarker Multicopy Reference Assay
The qBiomarker Copy Number Arrays have an integrated multi-copy reference assay for data normalization. The multi-copy reference assay  recognizes a stable sequence that appears in the human genome over 60 times, and whose copy number is not affected or minimally affected by local genomic changes. Inclusion of this reference assay during testing allows use of the ΔΔCT method to accurately make copy number calls or relative copy number change calls for specific targets.

 

qBiomarker Multicopy Reference Assay is superior to RNase P as a reference assay for copy number determination. Tumor cell line DNA (SKBR3) and reference genomic DNA were mixed in different ratios (100%, 87.5%, 75%, 50%, 25%, 12.5% and 0% SKBR3 cells respectively), and the DNA mixes were tested for GRB7 gene copy number, using either MRef assay or RNaseP assay as the reference. The GRB7 copy number in the reference genomic DNA is assumed to be 2. The "Expected" GRB7 gene copy numbers in 87.5%, 75%, 50%, 25%, 12.5% mixing ratio samples are calculated based on the GRB7 gene copy number in 100% SKBR3 genomic DNA sample, and the mixing ratio between the SKBR3 genomic DNA and Promega genomic DNA. The observed GRB7 gene copy numbers in general agree well with the expected values when using QIAGEN Multi-copy Reference Assay as the reference, while significant differences exist between the observed GRB7 gene copy numbers and the expected values when RNaseP is used as the reference assay.

RNaseP gene is not suitable as a normalizer of sample input in cancer cell line DNA samples. The absolute average copy numbers of RNaseP per normal genome copy amount of DNA were determined in two breast cancer cell line (SKBR3 and MCF7) genomic DNAs with the delta delta Ct method, using QIAGEN multi-copy reference assay as the normalization control of DNA input. The absolute copy number of RNaseP per normal genome in the genomic DNA  is assumed to be 2.

Universal Nature of Multicopy Reference Assay
The complete PCR Array System, with high quality input RNA, is guaranteed to yield single amplicons of the predicted size without primer dimers or other secondary products to ensure the most accurate real-time PCR results possible.

Stable Performance of the Multicopy Reference (MRef) Assay in 129 DNAs from 9 Major Human Populations. The qBiomarker multi-copy reference assay (MRef) and a qBiomarker copy number assay for RB1 were tested against DNAs from 129 healthy individuals from 9 major ethnic populations. Each assay and DNA sample combination was run in quadruple reactions. Delta CT between the average CT of the MRef assay and the RB1 assay was calculated for each individual DNA. The average delta Ct for samples within each ethnic population is plotted. Error bars show the standard deviation of the delta CT within each ethnic population. The RB1 gene is assumed to be present at 2 copies in all healthy individual DNAs.

Application Data

Aneuploidy Study

qBiomarker Copy Number Assays accurately identify aneuploidy. qBiomarker Copy Number Assays designed to target AR and MECP2 were tested against 4 cell line DNAs containing 1 copy (XY, Coriell NA13619), 2 copies (XX, Coriell NA01921), 3 copies (XXX, Coriell NA03623) and 4 copies (XXXX, Coriell NA11226) of X-chromosome. Chromosomal aberrations had been previously identified by cytogenetic methods. A control assay, targeting a stable, multi-copy region in the human genome, was used to normalize the amount of DNA input. ΔΔCT method was used to calculate the gene copy number, using XX (Coriell NA01921) as a 2-copy reference. Each assay was tested against each sample in quadruple replicate reactions, and a t-test was performed.