What is a Multicopy Reference Assay for copy number
Gene copy number measurements with qPCR require the use
of a normalization assay. The normalization assay is used to control for the
amount of input DNA. Traditionally, a normalization assay is designed against a
unique sequence in the genome such as a single copy gene. A multicopy reference
assay is designed against a repetitive sequence that is located at multiple
independent loci in the genome.
Why use a Multicopy Reference Assay?
Random Genomic Distribution
The qBiomarker Multicopy 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. By being evenly distributed
across the genome, amplifications or deletions of chromosomes are more
accurately factored into normalization calculations. This is in contrast to a
single gene normalization assay such as RNase P or TERT that only detect changes
to one location in a genome.
Random distribution of the qBiomarker Multicopy
Reference Assay. The individual chromosomes in the human genome are
displayed with arrows indicating the location of a repetitive sequence. The
qBiomarker Multicopy Reference Assay detects the presence of this sequence,
with no more than 10% coming from any single chromosome. Additional
criteria used for its selection included that when multiple loci are found
on the same chromosome that they occur on both arms.
nature of multicopy reference assay
A reference assay for genotyping must be independent of any influences
associated with various human populations. The qBiomarker Multicopy
Reference Assay is consistently present and detected across human
|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.
Single copy reference assays yield varying results
from unstable genomes
In cells with unstbale genomes, such as cancer cells,
amplifications and deletions of chromosomes quickly accumulate in a
semi-random manner. Therefore, single copy reference assays may not reflect
those changes accurately.
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 Promega genomic DNA (G304A) is assumed to be 2.
Multicopy reference assay better reflects input DNA
RNAseP as a reference assay
Multicopy Reference Assay
qBiomarker Multicopy Reference Assay is superior to
RNase P as a reference assay for copy number determination. Tumor cell
line DNA (SKBR3) and Promega genomic DNA (G304A) 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 PromegaŽ
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.
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