QIAcuity dPCR Copy Number Assays User Manual
Sample to Insight__
Quick-Start Protocol February 2021
dPCR Copy Number Assays
This protocol is optimized for the quantification of gene-specific regions in human DNA using the dPCR
Copy Number Assays (cat. nos. 250205, 250206) with the QIAcuity
®
250112, 250113), in an EvaGreen
detect copy number variation (CNV) in the human genome.
The dPCR Copy Number Assays are shipped on dry ice and should upon receipt be immediately stored
protected from light at –30 to –15°C in a constant-temperature freezer for up to 12 months or at 2–8°C
for up to 6 months. Under these conditions, the components are stable, without showing any reduction in
performance and quality, until the date indicated on the label.
-based reaction using the QIAcuity digital PCR (dPCR) instrument, to
Further information
QIAcuity User Manual Extension: QIAcuity Application Guide
QIAcuity User Manual
Safety Data Sheets: www.qiagen.com/safety
Technical assistance: support.qiagen.com
: www.qiagen.com/HB-2717
®
EG PCR Kit (cat. nos. 250111,
: www.qiagen.com/HB-2839
Notes before starting
A reference assay with known copy numbers/genome can be used to identify CNVs in test
samples, given that the same DNA sample is used across the reference and test assays.
Alternatively, a DNA sample with known copy number for gene of interest (or region of
interest) can be used as reference sample. We recommend that the quality and source of
reference sample(s) be comparable to that of test samples, if possible.
Reference assays and samples must be included in the same QIAcuity
®
Nanoplate, in
addition to test assays and samples. We recommend using multiple reference samples
and/or reference assays.
dPCR Copy Number Assays come as a 25x primer mix in a single tube.
Always start with the dPCR cycling conditions and primer concentrations specified in this
protocol.
2
dPCR Copy Number Assays 02/2021
Pipetting accuracy and precision affect the consistency of results. Make sure that no
bubbles are introduced into the wells of the QIAcuity Nanoplate during pipetting.
Due to the hot-start, it is not necessary to keep samples on ice during reaction setup or
while programming the QIAcuity instrument.
Template DNA digestion
Before partitioning, use restriction digestion to fragment DNA samples with average length
≥20 kb, to ensure even distribution of template throughout the QIAcuity Nanoplate, for
accurate and precise quantification.
DNA fragmentation via restriction digestion is critical for accurate CNV analysis when
multiple copies of a gene might be linked in tandem. Restriction digestion is not required for
highly fragmented DNA (e.g., FFPE DNA or circulating DNA) or cDNA.
To perform restriction digestion directly in reaction mix, add recommended restriction
enzyme during reaction setup. Use restriction enzymes that do not cut within target
amplicon regions.
We recommend using
Eco
RI-HF,
Pvu
II,
Xba
I (6-cutters),
Alu
I,
Cvi
QI,
Hae
III (4-cutters),
which are validated to digest template DNA in 10 min at RT in QIAcuity EG PCR Master
Mix without impairing the subsequent PCR amplification (Table 1). For additional assayspecific enzyme recommendations that do not cut in the amplicon, go to
geneglobe.qiagen.com or refer to the product data sheet (printout sent with the product).
Table 1. Validated restriction enzymes
6-cutter restriction enzymes 4-cutter restriction enzymes
Eco
RI 0.25 U/µl EcoRI-HF®, NEB®
0.025 U/µl Anza™ 11 EcoRI,
Thermo Fisher Scientific (TFS)
Pvu
II 0.025 U/µl PvuII, NEB
0.025 U/µl Anza 52 PvuII, TFS
Xba
I 0.025 U/µl Anza 12 XbaI, TFS
Procedure
Reaction setup
1. Thaw the QIAcuity EG PCR Master Mix, template DNA, dPCR Copy Number Assay, and
RNase-Free Water. Mix the individual solutions.
2. Prepare a reaction mix according to Table 2.
Alu
I 0.025 U/µl AluI, NEB
0.025 U/µl Anza 44 AluI, TFS
Cvi
QI 0.025 U/µl CviQI, NEB
0.025 U/µl Csp6I (CviQI), TFS
Hae
III 0.025 U/µl BsuRI (HaeIII), TFS
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