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Human Multicopy Reference PCR Assay

Human Mouse
 
Human Multicopy Reference PCR Assay
The qBiomarker Multicopy Reference Assay is used for the normalization of input DNA for both the qBiomarker Copy Number PCR Arrays and Assays. Normalization of input DNA for copy number analysis is critical for accurate measurements. While the addition of genomic DNA samples to a reaction mixture is one potential source of variability in input DNA, unstable genomes with large scale deletions and amplifications provide a greater challenge for accurate normalization. The use of a multicopy reference assay provides a more accurate measurement of input DNA by measuring loci evenly spread throughout the genome.

qBiomarker Multicopy Reference Assay benefits:

  • Superior normalization compared to single gene normalizers
  • Random genomic distribution yields reliable measurement of input DNA
  • Universal reagent with sequence stability across human populations

The qBiomarker Multicopy Reference Assay is included on all qBiomarker Copy Number PCR Arrays and is recommended for use with all qBiomarker Copy Number Assays.

The qBiomarker Copy Number PCR Assays and Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

 

How It Works Manual & Resources Reagents & Software
 

What is a Multicopy Reference Assay for copy number determination?

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.

Universal 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 populations.

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 amounts

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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How It Works Manual & Resources Reagents & Software
 
Handbook/User Manual qBiomarker Copy Number PCR Arrays and Assays (PDF)
Data Analysis Free Copy Number PCR Array Data Analysis Software (Web & Excel based)
Instrument Setup Instructions Materials & Equipment Required for our PCR products

PCR Array Instrument Setup Instructions & Protocol Files

FAQ FAQ for our Copy Number PCR products
Web Seminars Attend a live on-line seminar hosted by an Applications Scientist about PCR
Technical Brochures Copy Number PCR Array Brochure (PDF)
Powerpoint Presentations Copy Number Technology Overview Presentations
Pathway Central Pathway reviews and presentation-ready pathway maps
Individual Copy Number Assays Bench-Validated Copy Number qPCR assays for any locus in the genome
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How It Works Manual & Resources Reagents & Software
 

The complete qBiomarker Copy Number PCR Array System insures that you will have superior results from your pathway-focused copy number analysis experiment the first time, and every time guaranteed.

The Complete qBiomarker Copy Number PCR Array System Includes:

  • qBiomarker Copy Number PCR Arrays

  • qBiomarker SYBRŽ Green Mastermix
    Instrument-specific PCR master mixes insure high-efficiency, locus-specific amplification for the most accurate real-time PCR results.

  • qBiomarker Data Analysis Software (Excel & Web based)

Our convenient and high-quality PCR accessory products complete the qBiomarker PCR Array System and the qBiomarker Copy Number Assays

Other recommended accessory products:

QIAamp DNA Mini Kit (50)
Isolate genomic DNA from fresh or frozen samples for downstream real-time PCR array or assay analysis.

QIAamp DNA FFPE Tissue Kit
Isolate genomic DNA from fixed samples, including formalin-fixed paraffin-embedded (FFPE) blocks, paraffin blocks, sections, or slides, for downstream real-time PCR array or assay analysis.

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