Pancreatic Cancer qBiomarker Copy Number PCR Arrays

胰腺癌qBiomarker拷贝数PCR芯片
 

Product	Species	Technology	Cat. No.
Pancreatic Cancer qBiomarker Copy Number PCR Arrays	Human	Copy Number	VAHS-0054Z

The Human Pancreatic Cancer qBiomarker Copy Number PCR Array profiles the copy number of 23 genes reported to undergo frequent genomic alterations in human pancreatic tumor DNA. Pancreatic cancer, the fifth leading cause of cancer death, has a very poor prognosis upon diagnosis due to lack of any symptoms until the cancer reaches a late stage. Improving prognosis requires identifying early molecular markers, and progress has been made in identifying acquired copy number alterations in key oncogenic pathways. A large number of pancreatic tumors possess genetic alterations in signal transduction, transcription factor, and other genes that regulate apoptosis, cell cycle, differentiation, epigenetic modifications, and the Notch and TGFβ signaling pathways. Genes were chosen from the most frequently amplified or deleted genes relevant to oncogenic pathways and pancreatic cancer biology based on the primary literature and public databases. This array may serve as a useful tool to help classify pancreatic cancer samples by genotype and help verify pancreatic cancer phenotypic biomarkers. The array analyzes each gene in each sample in quadruplicate and includes a stable multi-copy reference assay for accurate copy number determination via appropriate DNA input normalization. The simplicity of the product format and operating procedure allow routine and reliable copy number profiling in any research laboratory with access to a real-time PCR instrument.
The qBiomarker Copy Number PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
 
胰腺癌qBiomarker拷贝数PCR芯片用于研究已报道的早胰腺肿瘤中发生频繁突变的23个基因的拷贝数。许多基因的拷贝数变异都会导致先天性异常。胰腺癌是癌症死亡的第五大原因,一旦诊断就已产生极不良的预后，由于缺乏任何症状,直到达到晚期癌症。改善预后需要识别早期的分子标记，这些程序已在关键致癌途径中确定拷贝数突变进行。大量的胰腺肿瘤在信号转导,转录因子,和其他基因调节细胞凋亡,细胞周期、分化、表观遗传修饰、Notch和TGFβ信号通路上具有遗传学改变。基于文献和数据库，该芯片上包含最频繁扩增或缺失的与致癌途径和胰腺癌生物学相关的基因。这个芯片可做为帮助分类胰腺癌样本的基因型并验证表型生物标记的有效工具来。这个芯片可以使每个基因在每个样本中有四个重复，同时包含一个稳定的多拷贝参考实验，通过适当的DNA插入标准化精确检测拷贝数。简单的产品模式和操作程序让任何一个具备实时定量PCR仪的实验室都可进行常规可靠的拷贝数检测。
qBiomarker拷贝数PCR芯片用于分子生物学应用。本产品不用于疾病的诊断、预防和治疗。
提供96孔板，384 -(4×96)板，和100 孔。
 
Cell Cycle:CDKN2B, E2F1.
Cytoskeleton Regulation:ARPC1A, PAK4.
Epigenetics:ARID1A, DNMT3B, MED29, TRRAP.
G-Protein Signaling:ARHGEF10.
Growth Factors:NOTCH4, TGFB3.
MAP Kinase Signaling:DUSP6.
Proteinase:CTSZ.
Transcripiton Factors:FOXA2, GATA6, NFATC2, RUNX1, RUNX3, TP73.
Ubiquitin Ligases:SMURF1.
Others:FEZ1, RAB4A, TUSC3.
 

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.