qBiomarker Copy Number PCR Arrays

For profiling copy number variations and alterations

Features

  • Focused copy number profiling for any real-time instrument
  • Wet bench-tested assays
  • Integrated multicopy reference assay for superior normalization
  • Complimentary, easy-to-use data analysis tool
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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 are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
✓ 24/7 automatic processing of online orders ✓ Knowledgeable and professional Product & Technical Support ✓ Fast and reliable (re)-ordering

Product Details

qBiomarker Copy Number PCR Arrays are designed to measure the number of copies for a panel of genomic loci that are associated with signaling pathways or diseases. These arrays detect either germline (copy number variations, or CNV) or acquired changes (copy number alterations, or CNA) in copy number. The genomic loci are selected from comprehensive curated databases, such as the Database of Genomic Variants (DGV), and literature reviews based on their clinical or functional relevance and frequency of occurrence.

Performance

 

When using real-time PCR to evaluate copy number changes in DNA samples, two elements are critical: real-time PCR assay performance, and reliable normalization of DNA input. Every qBiomarker Copy Number PCR Assay on a qBiomarker Copy Number PCR Array is 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.

Indeed, 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.

 

qBiomarker Copy Number PCR Arrays use the qBiomarker Multicopy Reference Copy Number PCR Assay (MRef) to provide superior normalization for DNA input. Single-copy reference genes such as RNase P can yield  unreliable normalization, as in cancer cell lines. By contrast, the MRef assay provides  accurate normalization, yielding more reliable results. Together, the laboratory verification of each qBiomarker Copy Number PCR Assay on the array and the superior DNA input normalization provided by the MRef assay ensure accurate, reliable results.

See figures

Principle

 

 

Each qBiomarker Copy Number PCR Array contains a panel of qBiomarker Copy Number PCR Assays for a stringently selected set of pathway- or disease-focused GOIs or ROIs. Four replicates of each assay are included to increase the accuracy of copy number calls using statistical analysis. The arrays are available in 96-well plate, 384-well plate, and Rotor-Disc formats. A  96-well plate array and a  Rotor-Disc array each contain 4 replicates of 24 genes (23 target genes plus MRef) for one sample, and a  384-well plate array can either contain 16 replicates of 24 genes (23 target genes plus MRef) for 4 samples, or 4 replicates of 96 genes (95 target genes plus MRef) for one sample.

 

All qBiomarker Copy Number PCR Assays are designed in unique regions of the genome. A multicopy reference assay, the qBiomarker Multicopy Reference Copy Number PCR Assay (MRef) is included on each array. The reference 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 ΔΔCT method to accurately make copy number calls or relative copy number change calls for specific targets.

The simplicity of the qBiomarker Copy Number PCR Array format and  operating procedure allows routine copy number profiling in any research laboratory with access to real-time PCR instruments.

 

 

See figures

Procedure

To complete the qBiomarker Copy Number PCR Array  procedure, start with genomic DNA isolated from fresh or frozen samples, or DNA from FFPE sections (QIAGEN QIAamp DNA Mini Kit or FFPE Tissue Kit is recommended). Optionally, if DNA sample quantity is limited, DNA from fresh or frozen tissues can be uniformly amplified using QIAGEN REPLI-g UltraFast Kit. Then, mix your DNA with the appropriate qBiomarker SYBR® Green Mastermix and aliquot the mixture into each well of the same qBiomarker Copy Number PCR Array plate containing predispensed locus-specific primer assays. Real-time PCR is used to determine the copy number status of a particular sample using the ΔΔCT method by comparing the test sample with a reference genome. An optional DNA sample quality control step can be performed immediately before the detection array setup.

 

 

See figures

Applications

qBiomarker Copy Number PCR Arrays are highly suited for accurate profiling of copy number alterations or variations in a pathway- or disease-focused set of genes.

Supporting data and figures

Resources

Instrument Technical Documents (2)
For gene expression and genomic analysis
For profiling copy number variation and alterations
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