Sample & Assay Technologies
QIAGEN webinar
Manual and automated large-volume extraction of
circulating nucleic acids
February 28, 2013
Dr. Martin Horlitz, Senior Scientist R&D
Sample & Assay Technologies
Regulatory Disclaimer
The applications presented here are for research use only.
Not for use in diagnostic applications.
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Extraction/isolation of circulating nucleic acids
Properties of circulating, cell-free nucleic acids found in
plasma and serum pose technical challenges
Low concentration compared to cells or tissue
as sample material (˂100 ng/ml plasma)
Biomarkers: Only a few molecules present per sample
Circulating DNA and RNA are highly fragmented
DNA: mostly shorter than
500–1000 bp
Fetal DNA: mostly shorter
than 500 bp
RNA: full-length mRNA is
rare, 5’ ends more intact
than 3’ ends
Need for optimized DNA/RNA extraction method
Process larger sample
volumes to isolate
sufficient absolute amounts
Allow for a low elution
volume to obtain high
concentrations
Manual and automated large-volume extraction of circulating nucleic acids
Efficiently recover small
DNA and RNA fragments
(down to 75 bp or lower)
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Sample & Assay Technologies
Example: circulating DNA — yield vs. enrichment
17%
Gel-separated circulating, cell-free DNA (ccfDNA) from 10 ml pooled maternal plasma
(1st and 2nd trimester):
 Total ccfDNA is present in plasma as fragments mostly <1000 bp
 Fetal ccfDNA is mostly shorter than 500 bp
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Example: circulating DNA — yield vs. enrichment
22%
17%
Gel-separated circulating, cell-free DNA (ccfDNA) from 10 ml pooled maternal plasma
(1st and 2nd trimester):
 Total ccfDNA is present in plasma as fragments mostly <1000 bp
 Fetal ccfDNA is mostly shorter than 500 bp
 Size-based enrichment: modest increase in % of fetal DNA, reduced absolute yield
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Importance of preanalytic solutions
Blood processing for ccfNA Analysis
Whole EDTA
blood
60 minutes or less
Spin at 1900 x g to
separate plasma
Carefully save
supernatant
Freeze below –20°C,
ship on dry ice,
store at –80°C
Spin at~16,000 x g
Reduced cellular gDNA background
Supernatant:
plasma w/o cell
debris
Extraction of
circulating, cell-free
DNA, RNA
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Solution: QIAamp® Circulating Nucleic Acid Kit
3 – 5 ml plasma,
serum, urine
Vacuum-based manual
processing of 24 samples
in 2–3 hours
Lysis
Add binding buffer to lysate
Binding
QIAamp column
on QIAvac 24 Plus
Wash steps
Elute: DNA/RNA
containing all
fragment sizes
in 20–150 µl volume
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Recovery of spike-in controls
Recovery of spiked DNA fragments from EDTA plasma — individual donors
 75 bp, 200 bp, 1000 bp DNA added to plasma before extraction (200,000 copies)
 Recovery measured by triplex qPCR (QuantiTect® Multiplex chemistry)

QIAamp Circulating Nucleic Acid Kit chemistry results in improved recovery of
DNA fragments shorter than 200 bp
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Benefit of large sample volumes
Circulating DNA in EDTA plasma — individual donors
 Circulating DNA yield: 18S rDNA qPCR — 66 bp and 500 bp amplicons
 Considerable sample-to-sample variability in ccfDNA yields

 Internal controls needed to track success of nucleic acid extraction
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Example: plasma circulating DNA and therascreen
QIAamp DNA
FFPE Tissue
Kit
CRC Tissue
(FFPE)
therascreen®
KRAS RGQ
Matched
plasma
QIAamp
Circulating
Nucleic Acid
Kit
Manual and automated large-volume extraction of circulating nucleic acids
PCR Kit
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Sample & Assay Technologies
Example: plasma circulating DNA and therascreen
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Example: fetal circulating nucleic acid purification
Study setup
 Collection of maternal plasma samples in cooperation with
Praenatal Medizin & Genetik, (Düsseldorf, Germany)
 Collection of 2 x 10 ml whole blood in EDTA tubes during 1st trimester
screening from 200 volunteers (12th – 13th week gestation stage)

Plasma separation on site within 60 minutes

Plasma frozen at –20°C

Transferred to QIAGEN labs on dry ice

Stored at –80°C until nucleic acid extraction

Before extraction: plasma centrifuged at 16,000 x g
 removal of cell debris, large chromatin fragments
 reduction of maternal gDNA background
 Extraction of circulating cell-free nucleic acids using commercially available
QIAGEN methods, analysis by qPCR (QuantiTect chemistry)
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Example: fetal circulating nucleic acid purification
QIAamp Circulating Nucleic Acid Kit
QIAamp DSP Virus Kit
Spin
columns
Sample volume: 5 ml
Elution volume: 70 µl
Sample volume: 500 µl
Elution volume: 70 µl
QIAsymphony® Virus Bacteria Midi
Kit Cell-free_1000 Protocol
EZ1® Virus Mini Kit v2.0
Magnetic
beads
Sample volume: 1 ml
Elution volume: 60 µl
Sample volume: 400 µl
Elution volume: 60 µl
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
qPCR assay details
Box plot (example)
Extracted circulating nucleic acids:
192 samples
x 4 extraction protocols
QIAsym
Virus
QIAamp
Circ.NA
QIAamp
DSP
EZ1
Virus 2.0
Maximum
90th percentile
ccfDNA: qPCR
18S
DYS14
75th percentile
Absolute
quantification
25th percentile
Genome
QuantiTect
chemistry
Spike-in DNA: qPCR
75 bp
200 bp
1000 bp
Median
10th percentile
Minimum
qPCR reactions were run in triplicates
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Total and fetal circulating DNA yields
Median fetal DNA
content: 15%
Similar fetal DNA
percentage using
different extraction
methods
method
Method
QIAsym Virus
median
no. of
of
Median
No.
GE/ sample valid results
1538
n = 184
QIAamp cN A
6882
n = 187
QIAamp DSP
917
n = 189
EZ1 Virus 2.0
482
n = 182
Automated and
manual extraction
methods perform
equally well
Manual and automated large-volume extraction of circulating nucleic acids
Method
method
median
no.ofof
Median
No.
GE/ sample valid results
QIAsym Virus
232
n = 82
QIAamp cN A
1141
n = 86
QIAamp DSP
138
n = 86
EZ1 Virus 2.0
76
n = 84
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Sample & Assay Technologies
Recovery of internal control DNA
No DNA extraction bias due to DNA
fragment length
 No loss of shorter fetal DNA
fragments
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
QIAsymphony circulating nucleic acid extraction
Larger sample volume | Virus cell-free 1000
1 ml plasma
Sample lysis
Binding to magnetic particles
Wash steps
Elution
Elute circulating DNA
<1000 bp
in <100 µl
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
QIAsymphony circulating nucleic acid extraction
Larger sample volume: customized protocol
4 ml plasma
Sample lysis
Binding to magnetic particles
4x lysis and binding
Wash steps
Elution
Elute circulating DNA
<1000 bp
in <100 µl
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Functional test of QIAsymphony 4 ml protocol
Extraction of circulating DNA using the QIAsymphony SP System
together with the QIAsymphony DSP Virus/Pathogen Midi Kit
Experimental setup:
 Plasma samples from 24 healthy donors
 Plasma separated from whole blood by double centrifugation
 Methods compared:

QIAamp Circulating Nucleic Acid Kit as manual
reference method
 4 ml plasma

QIAsymphony Virus/Pathogen Midi Kit with
custom 4 ml protocol
 4 ml plasma

QIAsymphony Virus/Pathogen Midi Kit with
custom 2 ml protocol
 2 ml plasma
 Circulating DNA yield determined by real-time PCR

18S coding sequence, 66 bp, and 500 bp amplicons

APP assay, 67 bp/180 bp/306 bp/475 bp
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Results — 18S qPCR assay: yield per ml plasma
18S coding sequence, 66 bp, and 500 bp amplicons
QIAamp Circulating NA Kit yield = 100%
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Results — 18S qPCR assay: comparable to QIAamp CNA
18S coding sequence, 66 bp, and 500 bp amplicons
QIAamp Circulating NA Kit yield = 100%
Comparable performance of QIAsymphony and QIAamp Circulating N.A. extractions
within the precision limits of qPCR
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Results — APP qPCR assay: comparable to QIAamp CNA
APP gene, 67 bp/180 bp/306 bp/475 bp amplicons
QIAamp Circulating NA Kit yield = 100%
Comparable performance of QIAsymphony and QIAamp Circulating N.A. extractions
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Circulating nucleic acids: analysis workflow
 Avoid release of cellular
nucleic acids
Blood draw
(venipuncture)
<1 hour
Separate plasma
 Highly efficient large-volume
nucleic acid extraction
Extract circulating nucleic acids:
QIAamp Circulating NA Kit,
QIAsymphony Virus/Pathogen Kit
 Maximize recovery
 improve sensitivity
Optional DNA modification
(e.g., bisulfite treatment)
Real-time PCR
digital PCR
therasceen assays
Sequencing library prep
Next-generation
sequencing
 Reduce interference of nontarget (“normal”) nucleic acids
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
Summary
Circulating nucleic acids in are highly fragmented and of very low
abundance  efficient extraction from plasma, serum, requires
optimized chemistry and easy processing of large volumes.
Circulating DNA from plasma enables cancer mutation analysis,
providing a “liquid biopsy” of a tumor.
QIAamp Circulating Nucleic Acid Kit (processing 5 ml plasma) allows:
 High yields and easy processing of large sample volumes
 High recovery of ccfDNA (75—1000 bp).
The QIAsymphony SP enables fully automated ccfDNA extraction —
including fetal DNA — from up to 4 ml plasma using the modified
QIAsymphony protocol.
Manual and automated large-volume extraction of circulating nucleic acids
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Sample & Assay Technologies
QIAGEN webinar
For up-to-date licensing information and product-specific disclaimers, see
the respective QIAGEN kit handbook or user manual. QIAGEN kit
handbooks and user manuals are available at www.qiagen.com or can be
requested from QIAGEN Technical Services or your local distributor.
Trademarks: QIAGEN®, QIAamp®, QIAsymphony®, EZ1®, QuantiTect®, therascreen® (QIAGEN Group).
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