qBiomarker Copy Number PCR Assays

For locus-specific analysis of copy number variations and alterations

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qBiomarker Copy Number PCR Assays

Cat. No. / ID: 337812

Laboratory-verified qPCR assays for measuring changes in copy number

qBiomarker Copy Number PCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Features

Over 10 million assays available, targeting all regions of the genome
Wet bench-tested assays
Simple real-time PCR procedure
Complimentary web-based data analysis Software

Product Details

qBiomarker Copy Number PCR Assays provide a method for obtaining specific, accurate, reproducible, and easy-to-interpret copy number change results for an individual gene or region of interest (GOI or ROI). Every assay has been bench-verified and is ready-to-use in microarray follow-up studies, specific target screening, and related studies.

Performance

All qBiomarker Copy Number PCR Assays are wet bench-tested for several characteristics affecting the accuracy of real-time PCR results: specificity, wide dynamic range, and uniformly high amplification efficiency. Laboratory verification of assay quality ensures that qBiomarker Copy Number PCR Assays deliver reliable results.

qBiomarker Copy Number PCR Assays accurately identified aneuploidy in cell lines containing chromosomal aberrations previously identified by cytogenetic methods. Assays for AR and MECP2, which are both on the X chromosome, correctly quantified gene copy number in cell lines with 1, 3, and 4 copies of the X chromosome.

See figures

qBiomarker Copy Number Assays accurately identify aneuploidy.

Principle

All qBiomarker Copy Number PCR Assays are designed in unique regions of the genome. The qBiomarker Multicopy Reference Copy Number PCR Assay (MRef), which can be purchased separately, provides superior normalization and is recommended for use with the qBiomarker Copy Number PCR Assays. The MRef assay recognizes a stable sequence that appears in the human genome over 40 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 ΔΔC_T method to accurately make copy number calls or relative copy number change calls for specific targets. The all-in-one, ready-to-use qBiomarker SYBR^® Green PCR Mastermixes are optimized to provide maximum locus-specific amplification with minimal primer dimers.

Procedure

Isolate genomic DNA from fresh, frozen, or FFPE samples. Combine an aliquot of each sample with the appropriate ready-to-use qBiomarker SYBR Green Mastermix and qBiomarker Copy Number PCR Assay. In another tube, mix an aliquot of each sample with the mastermix and the qBiomarker Multicopy Reference Copy Number PCR Assay. Run in a real-time cycler and determine copy number changes by the ΔΔC_T method by comparing the test sample with a reference genome using the complimentary data analysis tool.

Applications

qBiomarker Copy Number PCR Assays are highly suited for accurate detection of copy number alterations or variations at individual loci using fresh, frozen, or FFPE samples.

Supporting data and figures

qBiomarker Copy Number PCR Assays accurately identify aneuploidy.

Resources

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