Saskia Rehbaum, Nicole Mano, Martin Schlumpberger, Martin Horlitz, Georg Wieczorek QIAGEN Strasse 1, 40724 Hilden, Germany 1118968 09/2019 Isolation of Circulating Nucleic Acids for In Vitro Diagnostic Use Sample to Insight Introduction Circulating 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 Volumes Nucleic 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 C T 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 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 Binding Mixing 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% C T 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 ml Input volume 4 ml 5 ml 26 25 24 23 22 21 20 C T 1 ml 2 ml 3 ml Input volume 4 ml 5 ml Comparison of single-donor plasma (1 ml input). A qPCR. B Digital PCR (BioRad). 80,000 70,000 60,000 50,000 40,000 30,000 20,000 10,000 0 Copies/ml qPCR 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 Events ddPCR 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 Classic Protocol Lower limt Upper limit Breeze 35,000 30,000 25,000 20,000 15,000 10,000 5000 0 Copies/ml 5 ml sample input volume Classic Protocol Breeze 28 26 24 22 20 18 C T 1 ml sample input volume Classic Protocol Lower limt Upper limit Breeze 25 23 21 19 17 15 C T 5 ml sample input volume Classic Protocol Breeze cfDNA cfDNA cfRNA cfRNA