qBiomarker Copy Number PCR Arrays
For profiling copy number variations and alterations
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.
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.
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.
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.
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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