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FAQs : qBiomarker Copy Number PCR Arrays & Assays

Real-time PCR and PCR Array Technical FAQ
Q: What could have gone wrong if the CT values are unusually high for all wells in a sample?
A: One or more of the following 3 issues can lead to high CT values for all wells. (1) The cycling program is incorrect. Please make sure to program the real-time PCR cycler with the temperature profile shown in the protocols. (2) DNA quality is poor. Check the DNA quality using the DNA QC Plate (see the qBiomarker Copy Number PCR Array Handbook.) or on an agarose gel to see if the DNA is degraded. If the DNA is not degraded, it could be of insufficient purity. We recommend using one of the kits indicated in Table 3 of the qBiomarker Copy Number PCR Array Handbook for isolation of high-quality DNA. (3) Too little DNA is used. Make sure that the DNA has been properly quantified. During the DNA purification process, it is essential to perform an RNase digestion. RNA contamination in the DNA sample will lead to overestimation of DNA quantity. Note that larger amounts of DNA are recommended when working with FFPE samples.

Q: Why is the copy number call lower than expected?
A: Sample heterogeneity can lead to lower-than-expected copy number calls. In the presence of non-tumor cells that have normal diploid genomes, the copy number call is dependent on the copy number of the target gene in the cancer cells and the amount of non-tumor cells in the heterogeneous sample. If the percentage of non-tumor cells in the sample can be estimated, the copy number for a gene can be estimated based on Table 16 in the user manual.

Q: Why do qBiomarker Copy Number PCR Arrays have assays in quadruplicate?
A: Having the assays in quadruplicate will enable accurate copy number call via statistical analysis (implemented in online data analysis software).

Q: Can I use amplified genomic DNA with qBiomarker Copy Number PCR Arrays?

A: DNA from fresh frozen samples can be subjected to whole genome amplification (WGA) before use in downstream copy number PCR analysis. The recommended method is QIAGEN's REPLI-g or REPLI-g UltraFast Kits. For DNA from FFPE samples, we do not recommend amplification before copy number PCR analysis.

Q: Can I use genomic DNA from fixed samples with qBiomarker Copy Number PCR Arrays?
A: qBiomarker Copy Number PCR Arrays and Assays are compatible with fixed samples. However, when fixed samples are analyzed, the user is strongly recommended to refer to the "Important Points Before Starting" section and "Appendix A: Quality Control of Genomic DNA Using the DNA QC Plate" in the qBiomarker Copy Number PCR Array Handbook for considerations on selecting an appropriate calibrator sample(s), if available.

Q: How much DNA should I use on a qBiomarker Copy Number PCR Array?
A: Recommended amounts of genomic DNA per sample
Sample type   96-well and Rotorgene,
23-gene panel
384-well,
23-gene panel
384-well,
95-gene panel
Fresh tissue 0.4-1.0 µg 0.2-0.5 µg 0.8-2.0 µg
FFPE 0.8-1.2 µg 0.4-1.0 µg 1.6-4.0 µg

Q: How much DNA should I use in a qBiomarker Copy Number PCR Assay?
A:
Format 96-well plate or Rotor-Disc  384-well plate
Fresh tissue 4 ng 2 ng
FFPE 8 - 20 ng 4 - 10 ng

Q: Will qBiomarker Copy Number Assays work with heterogeneous samples like mixtures of tumor and normal tissue?
A: The assays will work with heterogeneous samples. However, because the tumor samples are "diluted" by the normal cells with diploid genome, the absolute observed copy number for each locus will be an average of the tumor cells and normal cells. However, the p-value should give an indication as to whether there is a statically significant amplification or deletion that happens in the cell population for the locus of interest.

Q: How do you choose which assays to include on the arrays?
A: In general, the genes on each qBiomarker Copy Number Array are selected from primary literature and public databases based on their amplification and deletion frequency in a disease or pathway, function in cancer or complex disease/trait signaling pathways, and their association with the disease phenotype or progression.

Q: How should I analyze the data generated from a qBiomarker Copy Number PCR Array experiment?
A: Data analysis uses the ΔΔCT method. At the qBiomarker Copy Number PCR Array and Assay Data Analysis Web portal (www.SABiosciences.com/dataanalysis.php), CT data can be entered and the Web-based software will automatically perform quantification.

Q: What qPCR mastermix should I use with the qBiomarker Copy Number PCR Arrays and Assays?
A: The qBiomarker SYBR ROX Mastermix is suitable for use with the following real-time cyclers: Applied Biosystems® models 5700, 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus™, ViiA™ 7 (Standard and Fast 96-well block, 384-well block); Eppendorf® Mastercycler® ep realplex models 2, 2S, 4, 4S; Stratagene® models Mx3000P®, Mx3005P®, Mx4000®; Takara TP-800.

The qBiomarker SYBR Fluor Mastermix is suitable for use with the following real-time cyclers: Bio-Rad® models iCycler®, iQ™5, MyiQ™, MyiQ2.

The qBiomarker SYBR ROX FAST Mastermix is suitable for use with the Applied Biosystems models 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus; ViiA 7 (Standard and Fast 96-well block, 384-well block); Eppendorf Mastercycler ep realplex with or without ROX filter set; Stratagene models Mx3000, Mx3500, Mx4000; Takara TP-800; Rotor-Gene® Q (QIAGEN), Rotor-Gene 6000.


Q: What sample types can I test on qBiomarker Copy Number PCR Arrays?
A: Various sample types can be used on the arrays, including fresh frozen cell line and tissue samples, cell line admixtures, PAXgene fixed tissue samples, and FFPE tissue samples.

Q: What testing should be performed in order to assess the quality of a DNA sample?
A: DNA concentration and purity can be measured by UV spectrophotometry

Dilute samples and measure absorbance in 10 mM Tris·Cl, pH 8.0. An absorbance reading of 1.0 at 260 nm in a 1 cm detection path corresponds to a DNA concentration of 50 µg/ml. All DNA samples should meet the following criteria:

  • Concentration, as measured by A260, should be greater than 10 µg/ml.
  • A260/A280 ratio should be greater than 1.8.
  • A260/A230 ratio should be greater than 1.7.

It is also strongly recommended that DNA quality be measured with the DNA QC Plate.

DNA quality and consistency can be checked more reliably with the DNA QC Plate by real-time PCR measuring 7 reference genes. For a detailed procedure, see the qBiomarker Copy Number PCR Array Handbook.

 
Q: Can I use DNA isolated from an AllPrep DNA/RNA Kit with qBiomarker Copy Number PCR Arrays and Assays?
A: Yes