Saskia Rehbaum, Nicole Mano, Martin Schlumpberger, Martin Horlitz, Georg WieczorekQIAGEN Strasse 1, 40724 Hilden, Germany

1118

968

0

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