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qBiomarker Copy Number PCR Arrays

For profiling copy number variations and alterations

  • 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

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.

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Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0001Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0002Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0052Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0053Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
show details
337802 VAHS-0054Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0055Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
show details
337802 VAHS-0056Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
show details
337802 VAHS-0057Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
show details
337802 VAHS-0058Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0502Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0503Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0504Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-0510Z varies
Panels of copy number assays in 96-well, 384-well, or Rotor-Disc formats
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337802 VAHS-3002Z varies
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.


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qBiomarker Copy Number PCR Arrays.

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RNase P and other single-copy genes are not suitable normalizers for sample input.
Genomic DNA samples with many amplifications and/or deletions, such as tumor DNA, require users to verify their single-copy reference genes to ensure that the reference genes themselves have not undergone a change in copy number. The advantage of a multi-copy reference assay is demonstrated in the measurement of the relative copy number of RNase P in 2 common cancer cell lines. The absolute average copy numbers of RNase P per normal genome copy amount of DNA were determined in 2 breast cancer cell line (SKBR3 and MCF7) genomic DNAs with the ΔΔCT method, using qBiomarker Multi-Copy Reference Copy Number PCR Assay as the normalization control of DNA input. The absolute copy number of RNase P per normal genome in reference genomic DNA is assumed to be 2. The copy number of RNase P in both SKBR3 and MCF7 is significantly altered, demonstrating that this reference gene would be an unreliable normalizer for these samples.
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MRef assay provides superior normalization for copy number determination.
Tumor cell line DNA (SKBR3) and reference genomic DNA (Promega) were mixed in different ratios (100%, 87.5%, 75%, 50%, 25%, 12.5%, and 0% SKBR3 cells), and the DNA mixes were tested for the gene copy number of GRB7 (a gene that is significantly amplified in SKBR3), using either the MRef assay (A) or the RNase P assay (B) as the reference. The GRB7 copy number in reference DNA is assumed to be 2. The expected GRB7 gene copy numbers in 87.5%, 75%, 50%, 25%, and 12.5% mixing ratio samples are calculated based on the GRB7 gene copy number in 100% SKBR3 gDNA sample, and the mixing ratio between the SKBR3 gDNA and reference genomic DNA.
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.

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.

 

 

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.

 

 

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.

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Handbooks & Protocols (1)
For profiling copy number variation and alterations
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Instrument Technical Documents (2)
For profiling copy number variation and alterations
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For gene expression and genomic analysis
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Kit Handbooks (1)
For real-time PCR-based copy number alteration and variation analysis
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