: qBiomarker Copy Number PCR Arrays & Assays
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
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
Q: Can I use amplified genomic DNA with qBiomarker Copy Number PCR
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
Q: How much DNA should I use on a qBiomarker Copy
Number PCR Array?
A: Recommended amounts of genomic DNA per sample
||96-well and Rotorgene,
Q: How much DNA should I use in a qBiomarker Copy
Number PCR Assay?
||96-well plate or Rotor-Disc
||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
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
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
- 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
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?