The Copy Number PCR array is a set of optimized real-time PCR primer assays on 96-well, 100-tube or 384-well plates for measuring changes in copy number. The PCR array performs copy number analysis with real-time PCR sensitivity and the multi-loci profiling capability of a microarray. Simply mix the genomic DNA sample with the appropriate ready-to-use PCR master mix, aliquot equal volumes to each well of the same plate, and then run the real-time PCR cycling program. (Download user manual)

The procedure involves isolating genomic DNA (QIAGEN QIAamp DNA Mini Kit or FFPE Tissue Kit is recommended), qPCR detection on qBiomarker Copy Number PCR Arrays or Assays, and data analysis (using the qBiomarker Copy Number Data Analysis). An optional DNA sample quality control step immediately before the detection array or assay setup allows the user to qualify the DNA samples.
To complete the Copy Number PCR Array procedure, start with 400 to1000 nanograms of genomic DNA isolated from fresh tissues, or as low as 800 nanograms of DNA from FFPE sections. Then, mix your DNA with the ready-to-use qBiomarker SYBR Green Mastermixes and aliquot the mixture into each well of the same plate containing pre-dispensed locus-specific primer assays. By performing real-time PCR, the copy number status of a particular sample is determined by comparing the CT values between your test sample and a wild-type control sample (see qBiomarker Copy Number Data Analysis section for detailed principles).

Why qBiomarker Copy Number Arrays?
     Guaranteed Performance* - ready-to-use for copy number analysis
     Time and cost saving - less than 30 minutes hands-on time for analyzing 23 loci
     Ease of data analysis using our easy-to-use Excel-based data analysis template or web-based analysis tool

Layout and Controls: The PCR Arrays are available in both 96-, 384-well plates and 100-well discs and are used to measure the copy number of 23 or 95 genes related to a disease state or pathway. Each gene/locus-specific assay is repeated in technical quadruplicate as indicated in the figure above. As an example, the first gene in the array is measured in wells A1, C1, E1 and G1. The qBiomarker Multicopy Reference Assay (MRef) is used to normalize the data for differences in the amounts of genomic DNA. The Multicopy Reference Assay is shown in purple in the above figure in wells B12, D12, F12, and H12.

You can easily perform a Copy Number PCR Array experiment in your own laboratory, or send your samples to us and take advantage of our PCR Array Services.

*:  when using qBiomarker SYBR? Green Mastermix.

Performance Data: 
The qBiomarker Copy Number PCR Array System yields a more accurate representation of changes in copy number by using a multi-copy reference assay to normalize the raw target specific data.

qBiomarker Multicopy Reference Assay
The qBiomarker Copy Number Arrays have an integrated multi-copy reference assay for data normalization. The multi-copy reference assay  recognizes a stable sequence that appears in the human genome over 60 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.

RNaseP gene is not suitable as a normalizer of sample input in cancer cell line DNA samples. The absolute average copy numbers of RNaseP per normal genome copy amount of DNA were determined in two breast cancer cell line (SKBR3 and MCF7) genomic DNAs with the delta delta Ct method, using QIAGEN multi-copy reference assay as the normalization control of DNA input. The absolute copy number of RNaseP per normal genome in the genomic DNA  is assumed to be 2.

Universal Nature of Multicopy Reference Assay
The complete PCR Array System, with high quality input RNA, is guaranteed to yield single amplicons of the predicted size without primer dimers or other secondary products to ensure the most accurate real-time PCR results possible.

Stable Performance of the Multicopy Reference (MRef) Assay in 129 DNAs from 9 Major Human Populations. The qBiomarker multi-copy reference assay (MRef) and a qBiomarker copy number assay for RB1 were tested against DNAs from 129 healthy individuals from 9 major ethnic populations. Each assay and DNA sample combination was run in quadruple reactions. Delta CT between the average CT of the MRef assay and the RB1 assay was calculated for each individual DNA. The average delta Ct for samples within each ethnic population is plotted. Error bars show the standard deviation of the delta CT within each ethnic population. The RB1 gene is assumed to be present at 2 copies in all healthy individual DNAs.

Application Data

qBiomarker Copy Number Assays accurately identify aneuploidy. qBiomarker Copy Number Assays designed to target AR and MECP2 were tested against 4 cell line DNAs containing 1 copy (XY, Coriell NA13619), 2 copies (XX, Coriell NA01921), 3 copies (XXX, Coriell NA03623) and 4 copies (XXXX, Coriell NA11226) of X-chromosome. Chromosomal aberrations had been previously identified by cytogenetic methods. A control assay, targeting a stable, multi-copy region in the human genome, was used to normalize the amount of DNA input. ΔΔCT method was used to calculate the gene copy number, using XX (Coriell NA01921) as a 2-copy reference. Each assay was tested against each sample in quadruple replicate reactions, and a t-test was performed.