qBiomarker Copy Number PCR Arrays

用于分析拷贝数变化和改变

S_1084_5_GEN_V2
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qBiomarker Copy Number PCR Arrays

Cat. No. / ID:  337802

Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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qBiomarker Copy Number PCR Arrays 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。
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Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Features

  • 在real-time PCR仪上进行拷贝数分析
  • 经湿法验证
  • 整合的多拷贝对照,用于标准化
  • 免费的即用型数据分析工具

Product Details

qBiomarker Copy Number PCR Arrays用于检测一组信号通路或疾病相关的基因组位点的拷贝数。芯片可检测生殖细胞(拷贝数变化或CNV)或获得性(拷贝数改变或CNA)的拷贝数变化。基因组位点是根据临床或功能相关性及发生频率,从全面的数据库和文献中挑选。

Performance

使用real-time PCR在DNA样本中进行拷贝数变化检测时,两个因素很关键:real-time PCR分析的表现和对DNA起始量的可靠标准化。在qBiomarker Copy Number PCR Array上的每个qBiomarker Copy Number PCR Assay都经湿法验证,有几个因素影响real-time PCR结果的准确性:特异性、宽动态范围和统一的高扩增效率。分析质量的实验室验证确保qBiomarker Copy Number PCR Assays获得可靠结果。

qBiomarker Copy Number PCR Assays可准确鉴定细胞学方法发现含有染色体畸变的细胞系中的非整倍体。检测X染色体上的 AR MECP2,可准备定量含有1、2、3、4拷贝X染色体细胞系的基因拷贝数。

qBiomarker Copy Number PCR Arrays使用qBiomarker Multicopy Reference Copy Number PCR Assay (MRef)对DNA起始量进行优良的标准化。单拷贝参照基因(如RNase P)在癌细胞系中会产生 不可靠的标准化。相比起来,MRef分析可提供 准确的标准化,产生更可靠地结果。总之,对芯片上的每个qBiomarker Copy Number PCR Assay的实验室验证,以及MRef分析提供的优良的DNA起始量标准化,可确保准确、可靠地结果。

See figures

Principle

每个qBiomarker Copy Number PCR Array含有一组qBiomarker Copy Number PCR Assays,分析一套严格筛选的的通路或疾病相关的GOIs或ROIs。每次分析都重复四次,以提高统计分析时拷贝数结果的准确性。芯片有96孔板、384孔板和Rotor-Disc规格。 96孔板芯片 Rotor-Disc芯片各含有一个样本24个基因(23个靶基因和MRef)的4次重复,或是一个样本96个基因(95个靶基因和MRef)的4次重复。

所有qBiomarker Copy Number PCR Assays都设计在基因组的独特区域。每个芯片都含有一个多拷贝参照分析,即qBiomarker Multicopy Reference Copy Number PCR Assay (MRef)。参照分析检测人体基因组中出现超过40次的稳定序列,其拷贝数不会受自身基因组变化影响或影响很小。检测包含参照分析可运用ΔΔCT法对特定靶标进行准确的拷贝数分析或相对拷贝数变化分析。

qBiomarker Copy Number PCR Array的规格和 操作流程的简便性使常规的拷贝数分析可在任何有real-time PCR仪的实验室进行。

See figures

Procedure

完成qBiomarker Copy Number PCR Array 操作流程的第一步是从新鲜或冷冻样本中分离基因组DNA,或是从福尔马林固定石蜡包埋切片中提取DNA(推荐使用QIAGEN QIAamp DNA Mini Kit或FFPE Tissue Kit)。如果DNA样本量很少,从新鲜或冷冻组织中提取的DNA可使用QIAGEN REPLI-g UltraFast Kit统一扩增。将DNA与合适的qBiomarker SYBR® Green Mastermix混合后,等体积加入到同一qBiomarker Copy Number PCR Array孔板的每个孔中,孔板含有预分配的位点特异性引物分析。使用real-time PCR确定特定样本的拷贝数状态,使用ΔΔCT法将检测样本与参照基因组对比进行分析。在芯片体系构建之前可选择性进行DNA样本质量控制步骤。
See figures

Applications

qBiomarker Copy Number PCR Arrays非常适合准确分析一套通路或疾病相关基因的拷贝数改变或变化。

Supporting data and figures

Resources

安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
仪器技术参数 (2)
For gene expression and genomic analysis
For profiling copy number variation and alterations
试剂盒操作手册 (1)
For real-time PCR-based, copy number alteration and variation analysis
Safety Data Sheets (1)
Certificates of Analysis (1)
Instrument Technical Documents (2)
For profiling copy number variation and alterations
For gene expression and genomic analysis
Kit Handbooks (1)
For real-time PCR-based, copy number alteration and variation analysis

FAQ

What sample types can I test on qBiomarker Copy Number PCR Arrays?
Various sample types can be used on the arrays, including fresh frozen cell line and tissue samples, cell line admixtures, PAXgene fixed tissue samples, and FFPE tissue samples.
FAQ ID — 3423
How much DNA should I use on a qBiomarker Copy Number PCR Array?
Recommended amounts of genomic DNA per sample
Sample type 96-well and Rotor-Gene,
23-gene panel
 384-well,
23-gene panel
 384-well,
95-gene panel
Fresh tissue 0.4–1.0 µg  0.2–0.5 µg  0.8–2.0 µg
FFPE 0.8–1.2 µg  0.4–1.0 µg  1.6–4.0 µg
FAQ ID — 3417
Will qBiomarker Copy Number Assays work with heterogeneous samples like mixtures of tumor and normal tissue?
The assays will work with heterogeneous samples. However, because the tumor samples are “diluted” by the normal cells with diploid genome, the absolute observed copy number for each locus will be an average of the tumor cells and normal cells. However, the p-value should give an indication as to whether there is a statistically significant amplification or deletion that happens in the cell population for the locus of interest.
FAQ ID — 3419
Can I use genomic DNA from fixed samples with qBiomarker Copy Number PCR Arrays?
qBiomarker Copy Number PCR Arrays and Assays are compatible with fixed samples. However, when fixed samples are analyzed, the user is strongly recommended to refer to the “Important Points Before Starting” section and “Appendix A: Quality Control of Genomic DNA Using the DNA QC Plate” in the qBiomarker Copy Number PCR Array Handbook for considerations on selecting an appropriate calibrator sample(s), if available.
FAQ ID — 3416
Why do qBiomarker Copy Number PCR Arrays have assays in quadruplicate?
Having the assays in quadruplicate will enable accurate copy number call via statistical analysis (implemented in online data analysis software).
FAQ ID — 3414
What qPCR mastermix should I use with the qBiomarker Copy Number PCR Arrays and Assays
The qBiomarker SYBR ROX Mastermix is suitable for use with the following real-time cyclers: Applied Biosystems models 5700, 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus, ViiA 7 (Standard and Fast 96-well block, 384-well block); Eppendorf Mastercycler ep realplex models 2, 2S, 4, 4S; Stratagene models Mx3000P, Mx3005P, Mx4000; Takara TP-800.

The qBiomarker SYBR Fluor Mastermix is suitable for use with the following real-time cyclers: Bio-Rad models iCycler, iQ5, MyiQ, MyiQ2.

The qBiomarker SYBR ROX FAST Mastermix is suitable for use with the Applied Biosystems models 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus; ViiA 7 (Standard and Fast 96-well block, 384-well block); Eppendorf Mastercycler ep realplex with or without ROX filter set; Stratagene models Mx3000, Mx3500, Mx4000; Takara TP-800; Rotor-Gene Q (QIAGEN), and Rotor-Gene 6000.

FAQ ID — 3422
Can I use DNA isolated from an AllPrep DNA/RNA Kit with qBiomarker Copy Number PCR Arrays and Assays
Yes.
FAQ ID — 3425
How should I analyze the data generated from a qBiomarker Copy Number PCR Array experiment?
Data analysis uses the ΔΔCT method. At the qBiomarker Copy Number PCR Array and Assay Data Analysis Web portal (http://www.qiagen.com/products/genes and pathways/data analysis center overview page), CT data can be entered and the Web-based software will automatically perform quantification.
FAQ ID — 3421
Why is the copy number call lower than expected?
Sample heterogeneity can lead to lower-than-expected copy number calls. In the presence of non-tumor cells that have normal diploid genomes, the copy number call is dependent on the copy number of the target gene in the cancer cells and the amount of non-tumor cells in the heterogeneous sample. If the percentage of non-tumor cells in the sample can be estimated, the copy number for a gene can be estimated based on Table 16 in the user manual.
FAQ ID — 3413
What could have gone wrong if the CT values are unusually high for all wells in a sample?
One or more of the following 3 issues can lead to high CT values for all wells:

1. The cycling program is incorrect. Please make sure to program the real-time PCR cycler with the temperature profile shown in the protocols.

2. DNA quality is poor. Check the DNA quality using the DNA QC Plate (see the qBiomarker Copy Number PCR Array Handbook) or on an agarose gel to see if the DNA is degraded. If the DNA is not degraded, it could be of insufficient purity. We recommend using one of the kits indicated in Table 3 of the qBiomarker Copy Number PCR Array Handbook for isolation of high-quality DNA.

3. Too little DNA is used. Make sure that the DNA has been properly quantified. During the DNA purification process, it is essential to perform an RNase digestion. RNA contamination in the DNA sample will lead to overestimation of DNA quantity. Note that larger amounts of DNA are recommended when working with FFPE samples.
FAQ ID — 3412
What testing should be performed in order to assess the quality of a DNA sample?
DNA concentration and purity can be measured by UV spectrophotometry. 

Dilute samples and measure absorbance in 10 mM Tris•Cl, pH 8.0. An absorbance reading of 1.0 at 260 nm in a 1 cm detection path corresponds to a DNA concentration of 50 µg/ml. All DNA samples should meet the following criteria:

1. Concentration, as measured by A260, should be greater than 10 µg/ml
2. A260/A280 ratio should be greater than 1.8
3. A260/A230 ratio should be greater than 1.7

It is also strongly recommended that DNA quality be measured with the DNA QC Plate.
DNA quality and consistency can be checked more reliably with the DNA QC Plate by real-time PCR measuring 7 reference genes. For a detailed procedure, see the qBiomarker Copy Number PCR Array Handbook.

FAQ ID — 3424
How much DNA should I use in a qBiomarker Copy Number PCR Assay?
Header
Format Genomic DNA
96-well plate / Rotor-Disc (per reaction) 4 ng (fresh); 8–20 ng (FFPE)
384-well plate (per reaction) 2 ng (fresh); 4–10 ng (FFPE)

FAQ ID — 3418
Can I use amplified genomic DNA with qBiomarker Copy Number PCR Arrays?
DNA from fresh frozen samples can be subjected to whole genome amplification (WGA) before use in downstream copy number PCR analysis. The recommended method is QIAGEN’s REPLI-g or REPLI-g UltraFast Kits. For DNA from FFPE samples, we do not recommend amplification before copy number PCR analysis.
FAQ ID — 3415
How do you choose which assays to include on the arrays?
In general, the genes on each qBiomarker Copy Number Array are selected from primary literature and public databases based on their amplification and deletion frequency in a disease or pathway, function in cancer or complex disease/trait signaling pathways, and their association with the disease phenotype or progression.
FAQ ID — 3420