qBiomarker Copy Number PCR Assays

コピー数多型と変化を遺伝子座特異的に解析

GeneGlobeで探索・設定する

適切なターゲット特異的アッセイおよびパネルを探すか、またはターゲットをカスタムデザインし、お客様のご興味のある生物学的ターゲット評価にお使いください。

qBiomarker Copy Number PCR Assays

Cat. No. / ID: 337812

Laboratory-verified qPCR assays for measuring changes in copy number

GeneGlobeで探索・設定する価格を確認するには

GeneGlobeで探索・設定する

qBiomarker Copy Number PCR Assaysは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

GeneGlobeで探索・設定する

特徴

ゲノム全領域をターゲットする1000万以上のアッセイが入手可能
ウエットベンチによる試験済みアッセイ
簡単なリアルタイムPCR手法
ウェブベースの無料のデータ解析ソフトウェア

製品詳細

qBiomarker Copy Number PCR Assaysは、個別の目的遺伝子（GOI）または目的領域(ROI)において特異的で、正確かつ再現性があり、解析しやすいコピー数変化結果を獲得する方法を提供します。すべてのアッセイは実験室で検証済みで、マイクロアレイのフォローアップ研究、特定ターゲットのスクリーニング、および関連研究ですぐに使用できます。

パフォーマンス

qBiomarker Copy Number PCR Assaysはすべて、リアルタイムPCR結果の正確さに影響する次のようないくつかの特性についてウエットベンチで試験済みです：特異性、広いダイナミック・レンジ、均一で高い増幅効率。アッセイ品質のラボ検証により、qBiomarker Copy Number PCR Assaysで信頼性の高い結果が確実に得られます。

qBiomarker Copy Number PCR Assaysは、すでに細胞遺伝学的方法によって同定された染色体異常を含む細胞株において、異数性を正確に同定しました。X染色体上に共に存在する AR と MECP2のアッセイは、1、3、4コピーのX染色体を持つ細胞株での遺伝子コピー数を正確に定量しました。

図参照

qBiomarker Copy Number Assays accurately identify aneuploidy.

原理

すべてのqBiomarker Copy Number PCR Assayは、独自のゲノム領域内に設定されています。qBiomarker Multicopy Reference Copy Number PCR Assay（MRef、別途購入可能）は、DNAインプットの卓越した正規化を実現し、qBiomarker Copy Number PCR Assaysと併用することを推奨します。MRefアッセイは、ヒトゲノムに40回以上存在する安定した配列を認識し、そのコピー数は局所的なゲノム変化の影響を全く、あるいは殆ど受けません。テスト中にこのリファレンスアッセイを同時に実施することにより、ΔΔC_T法を用いて、特定のターゲットに対してコピー数のコールあるいは相対的なコピー数変化のコールを正確に行なえます。オールインワンタイプで即使用可能なqBiomarker SYBR^® Green PCR Mastermixesは、最小限のプライマーダイマーで遺伝子座特異的な最大増幅を提供するために最適化されています。

操作手順

新鮮、凍結、FFPEサンプルからのゲノムDNAを単離します。各サンプルの溶液を、適切な即使用可能なqBiomarker SYBR Green MastermixおよびqBiomarker Copy Number PCR Assayと混和します。別のチューブに、一定量の各サンプルとマスターミックスならびにqBiomarker Multicopy Reference Copy Number PCR Assayを混和します。リアルタイムサイクラーを実行し、無料のデータ解析ツールを用いて、テストサンプルとリファレンスゲノムを比較するΔΔC_T法により、コピー数の変化を計算します。

アプリケーション

qBiomarker Copy Number PCR Assaysは、新鮮、凍結、FFPEサンプルから、個別の遺伝子座におけるコピー数異常や多型の正確な検出に最適です。

裏付けデータと数値

qBiomarker Copy Number PCR Assays accurately identify aneuploidy.

リソース

Download Safety Data Sheets for QIAGEN product components.

For real-time PCR-based, copy number alteration and variation analysis

For profiling copy number variation and alterations

FAQ

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

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

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

Having the assays in quadruplicate will enable accurate copy number call via statistical analysis (implemented in online data analysis software).

FAQ ID — 3414

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

Yes.

FAQ ID — 3425

Data analysis uses the ΔΔC_T 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), C_T data can be entered and the Web-based software will automatically perform quantification.

FAQ ID — 3421

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

One or more of the following 3 issues can lead to high C_T 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

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

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

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

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