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Custom qBiomarker Copy Number PCR Array

Custom PCR Arrays Tailored to Your Research
We make custom PCR Arrays containing assays for any combination of human genomic assays. Our genome-wide coverage provides assays for every 200 base pairs covering the entire Human genome. Custom PCR Arrays available in 96-well Plate, 384-well Plate, and 100-Well Disc

Why Custom qBiomarker Copy Number PCR Arrays?

  • Validate SNP array or aCGH results
  • Fine Map Deletion breakpoints or Gene Amplifications
  • Profile non-overlapping DGVs for individual genes
  • Streamline testing large numbers of by reducing liquid handling

 
Plate Layouts QA/QC Parameters Controls & Data Analysis
 

How to Order:

  1. Download the Custom Array Design File
  2. Select a plate layout to match the gene number and the number of samples you would like to analyze.
    • Please have the type of real time thermocycler available when placing the order.
    • (Optional) Custom Arrays can be used in our Service Core
  3. Determine the number and types of plates required.
  4. Use the Custom Array Design File to submit your assay list.
    • Please supply the current Assay ID for each genomic tile that you want on the array
    • To find the Assay ID for each locus, please visit: qBiomarker Copy Number Assay Search 
      or
    • Please submit a list of Gene ID or ReSeq #s and we will identify a single assay per gene
  5. Contact Technical Support at 1-888-503-3187 to submit your file and obtain a quote.
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Plate Layouts QA/QC Parameters Controls & Data Analysis
  Quality Assurance / Quality Control (QA/QC) Parameters

Primer Quality Control (QC)
Any algorithm's primer design must also be experimentally validated for high-performance with wet bench quality control protocols starting with two major success criteria.

  • First, a melt curve analysis must verify that a single gene-specific product is produced. Following the melt curve, an agarose gel can also be run to further verify a single product of the predicted size, based on the amplicon design, without primer dimers or off-target amplifications.
  • Second, the amplification efficiency must be greater than 90 percent for accurate and reliable results. If a real-time RT-PCR assay does not meet all of the above requirements, then the quality control fails, and the assay must be re-designed.

QA/QC Specificity

All real-time PCR assays must generate a single band of the correct size for the results to accurately represent the expression of the queried gene. Secondary products confound the analysis. If using SYBR Green-based detection, you can tell if your real-time PCR assays are specific enough by simply running the default melting program on your instrument immediately after the completion of the cycling program. A single peak indicates a single melting event, and therefore a single product.

Efficiency

Of the various methods of determining amplification efficiency, the most rigorous and classical method examines the slope of a calibration curve, much like those used to assess dynamic range. An assay

Primer Design Algorithm

Primer design algorithm is key to effective qPCR based gene expression analysis. Designs must meet several important thermodynamic and sequence criteria. Primers are designed such that they must detect every alternative transcript and splicing variant of the queried gene so as not to miss any genes.

Amplicon Length  50-150 base pairs
Primer Length  19-25 nucleotides
GC Content  35-65 %
Tm  60-68 ºC
3'-End Stability Composition of last 3 base pairs
Complementaries  Avoid primer self or cross- annealing stretches >4 base pairs
Specificity BLAST against human genome build 37
SNP Database Primer sequences do not include SNP with >0.01 Average Heterozygosity

To do so, all known entries in the public databases should be found and aligned to reveal a common gene-specific region for primer design.

  • By controlling the GC content, primer length, and the primer melting temperature range, each assay can use a standard set of PCR cycling conditions.

  • Uniform cycling conditions, in turn, allow researchers to scale up from a single assay, to multiple assays on an entire 96- or even 384-well plate.

  • Single Nucleotide Polymorphism (SNP) analysis can eliminate repetitive sequences so that any individual source of total RNA may be analyzed with the same assay.

  • BLAST analysis further insures that the chosen primer sequences are sufficiently different from the rest of the transcriptome in the species of interest.

  • Stability at the 3'-end of the primers controls the start position for the DNA polymerase, further enhancing specificity.

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Plate Layouts QA/QC Parameters Controls & Data Analysis
 
Recommended qBiomarker Copy Number PCR Array Control Element
Many factors are known to influence real-time PCR assay analyses, even if the performance of the assay itself is validated and optimized-from input DNA quality, PCR cycling conditions, to the performance of real-time PCR instruments. For data normalization purposes, we recommend users include the qBiomarker Multi-copy Reference Assay into each custom-made qBiomarker Copy Number PCR array.

Data Analysis
All Custom qBiomarker Copy Number Arrays are compatible with the qBiomarker Copy Number PCR Array Data Analysis tools.

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