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"If you have access to a real-time PCR instrument, you can use
PCR Arrays"
qBiomarker Copy Number PCR Arrays are the most reliable
and sensitive copy number profiling technology for analyzing a panel of loci in
signal transduction pathways or disease related gene networks. The Copy Number
PCR Arrays can be used for research on cancer, neurobiology, immunology,
biomarker discovery and validation. See the complete list of Copy Number PCR Arrays.
You can use any 96-well, 384-well plate or 100-well disc real-time PCR instrument. See
instrument-specific setup instructions.
How it Works
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)
| Figure 1: |
How Copy Number PCR Arrays Work - Protocol Chart |
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 SYBRŪ 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.
| qBiomarker Multicopy Reference Assay is
superior to RNase P as a reference assay for copy number determination.
Tumor cell line DNA (SKBR3) and reference genomic DNA were mixed
in different ratios (100%, 87.5%, 75%, 50%, 25%, 12.5% and 0% SKBR3
cells respectively), and the DNA mixes were tested for GRB7 gene copy
number, using either MRef assay or RNaseP assay as the reference. The
GRB7 copy number in the reference genomic DNA is assumed to be 2. The
"Expected" GRB7 gene copy numbers in 87.5%, 75%, 50%, 25%,
12.5% mixing ratio samples are calculated based on the GRB7 gene copy
number in 100% SKBR3 genomic DNA sample, and the mixing ratio between
the SKBR3 genomic DNA and Promega genomic DNA. The observed GRB7 gene
copy numbers in general agree well with the expected values when using
QIAGEN Multi-copy Reference Assay as the reference, while significant
differences exist between the observed GRB7 gene copy numbers and the
expected values when RNaseP is used as the reference assay. |
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
Aneuploidy Study
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.
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Complete Array List
