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Saskia Rehbaum, Nicole Mano, Martin Schlumpberger, Martin Horlitz, Georg WieczorekQIAGEN Strasse 1, 40724 Hilden, Germany
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9/20
19
Isolation of Circulating Nucleic Acids for In Vitro Diagnostic Use
Sample to Insight
IntroductionCirculating nucleic acids play an increasing role in obtaining diagnostic information in non-invasive prenatal testing (NIPT)
and in non-invasive analysis of the genetic makeup of tumors (liquid biopsy). The performance of such assays is dependent
on the efficient and complete extraction of circulating nucleic acids from blood samples.
We recently developed a protocol for the industry-leading QIAamp® DSP Circulating Nucleic Acid Kit specifically for in
vitro diagnostic use (Classic protocol). The protocol uses proven silica-membrane technology for isolation and purification
of circulating, cell-free DNA and RNA from human blood plasma samples.
To improve the usability and processing time of the extraction process, we also developed an optional, streamlined version
of the circulating nucleic acid isolation protocol that reduces processing steps and time (Breeze protocol).
Data show equivalent performance and excellent reproducibility. Furthermore, performance of eluates in different
downstream applications is fully maintained with the Breeze protocol.
Protocols in the QIAamp Circulating NA Kit:
• The Classic protocol is currently deployed in the kit and remains an option.
• The new Breeze protocol is optimized for faster turnaround.
Workflow and Comparison of Classic and Breeze Protocols
cfDNA/cfRNA Results with Different Plasma Input VolumesNucleic acids were isolated from 1–5 ml of human blood plasma using the new Breeze protocol of the QIAamp DSP
Circulating NA Kit. A validated qPCR assay targeting the human ribosomal DNA gene was used to detect cell-free DNA,
while a reverse transcription qPCR assay for the human beta-actin gene was used to detect cell-free RNA.
Precision evaluation for the QIAamp DSP Circulating NA Kit with regard to run-to-run (n=12), operator (n=3), instrument
(n=3), day-to-day (n=3) and lot-to-lot (n=3) reproducibility.
Results: Protocol Equivalency
Conclusions• The kit can be used in diagnostic workflows with human plasma as the sample material
• Isolated cfDNA and cfRNA are ready to use in downstream applications, such as qPCR, RT-PCR, digital PCR and
NGS (data not shown)
• A new protocol (Breeze) has been developed with shorter hands-on and turnaround times. The protocol shows equivalent
performance to the current (Classic) protocol
• The QIAamp DSP Circulating NA Kit shows linearity with regard to plasma volume input and a high reproducibility
between kit lots, runs, operators and instruments used
The QIAamp DSP Circulating NA Kit is intended for in vitro diagnostic use.
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®, Sample to Insight®, QIAamp® (QIAGEN Group). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.© 2019 QIAGEN, all rights reserved. PROM-14884-001
Comparison of dPCR with qPCR
Reproducibility of Circulating Nucleic Acid Isolation
QIAamp DSP Circulating Nucleic Acid Kit workflow. Spin column-based workflow for isolation of cell-free nucleic acids (DNA/RNA) from 1–5 ml human blood plasma using a vacuum procedure.The QIAamp DSP Circulating Nucleic Acid Kit.
Plasma cell-free DNA recovery for different input volumes using the Breeze protocol. Linear increase of cell-free DNA yield with increase of plasma input volume as detected by qPCR targeting the human rDNA gene (66 bp assay; constant elution and template volume).
Plasma cell-free RNA recovery for different input volumes using the Breeze protocol. Decrease of CT values with increasing plasma input volume detected by RT-qPCR targeting the human beta-actin gene (293 bp assay; constant elution and template volume).
Equivalent detection of cfDNA and cfRNA using Classic and Breeze protocols. Nucleic acids were isolated from 1 ml and 5 ml of human blood plasma using the Classic (current) and new Breeze (short) protocol of the QIAamp DSP Circulating NA Kit to show equivalency for cfDNA and cfRNA isolation from human plasma. A For detection of cell-free DNA, a validated qPCR assay targeting the human ribosomal DNA gene (66 bp fragment length) was used. B For cell-free RNA, a reverse transcription-qPCR assay for the human beta-actin gene (293bp fragment length) was used.
Sample
Lyse
QIAamp DSP Circulation NA Kit Procedure
Bind
Pure nucleic acids
Elute
Wash
Vacuum
Vacuum
Comparison of Classic and Breeze workflows
Comparison of hands-on time
Protocol step Classic protocol Breeze protocol
Lysis
Mixing conditions Pulse-vortex (30s) vortex (5 x 2s)
Temperature 60°C 56°C
Incubation time 30 min 15 min
BindingMixing conditions Pulse-vortex (15–30s) Vortex (5 x 2s)
Incubation temperature and time 5 min on ice 5 min at room
temperature
Elution Drying step 10 min at 56°C (heat block)
3 min at room temperature
Classic protocol Breeze protocol
~ 90 min ~ 50 min
Analysis variable
No.
amplified
No. not
amplified
Mean
Between run
(SD, %CV)
Between operator (SD, %CV)
Between instrument (SD, %CV)
Between day
(SD, %CV)
Between kit lot
(SD, %CV)
Residual
(SD, %CV)
Total number (SD, %CV)
Copies/ml 143 0 25,894.41 461.17, 1.78%
1392.29, 5.38%
228.35, 0.88%
2095.71, 8.09%
968.58, 3.74%
2429.11, 9.38%
3120.30, 12.05%
CT value 143 0 21.09 0.0417, 0.20%
0.0707, 0.34%
0.0000, 0.00%
0.257, 0.12%
0.0566, 0.27%
0.1133, 0.54%
0.1387, 0.66%
Variance components reported in terms of standard deviation (SD) and percentage coefficient of variation (%CV)
120,000
100,000
80,000
60,000
40,000
20,000
0
Copies/eluate
1 ml 2 ml 3 mlInput volume
4 ml 5 ml
26
25
24
23
22
21
20
CT
1 ml 2 ml 3 mlInput volume
4 ml 5 ml
Comparison of single-donor plasma (1 ml input). A qPCR. B Digital PCR (BioRad).
80,00070,00060,00050,00040,00030,00020,00010,000
0
Copies/mlqPCR ETDA plasma
1 2 3
Donor.4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1,400,000
1,200,000
1,000,000
800,000
600,000
400,000
200,000
0
EventsddPCR ETDA plasma
1 2 3
Donor.4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
45,000
35,000
25,000
15,000
5000
-5000
Copies/ml
1 ml sample input volume
ClassicProtocol
Lower limtUpper limit
Breeze
35,00030,00025,00020,00015,00010,000
50000
Copies/ml
5 ml sample input volume
ClassicProtocol
Breeze
28
26
24
22
20
18
CT
1 ml sample input volume
ClassicProtocol
Lower limtUpper limit
Breeze
25
23
21
19
17
15
CT
5 ml sample input volume
ClassicProtocol
Breeze
cfDNA
cfDNA cfRNA
cfRNA