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cfDNA isolation from plasma

High yields with chemagic™ auto­mated or manual puri­fica­tion of cir­cu­la­ting cell free DNA

Circulating cell free DNA (cfDNA) in body fluids is being used for noninvasive, real-time biomarker research and more. Yet detection is often challenged by its low levels, fragmentation and preanalytical variabilities introduced by sample collection, plasma preparation, and nucleic acid purification. 

Revvity chemagic cfDNA extraction kits, together with dedicated chemagic instruments rely on patented chemagic technology using M-PVA Magnetic beads to ensure a high quality of nucleic acid purification. Comparable yields to manual spin column methods were obtained with efficient removal of contaminants and exclusion of cross-contamination. With automation, variabilities associated with multiple handling steps are reduced and sample integrity maintained with full sample tracking capabilities.

Sprungziel Features

Features

Benefits of automated cfDNA isolation with chemagicTM technology:

  • Reliable and comparable yields to manual methods
  • Fast, automated workflow with on-board lysis and no heating required
  • Wide range of sample inputs applicable from 0.5 to 18 ml
  • Full sample tracking with barcode reading and bidirectional LIMS communication capability
  • Ability to isolate viral nucleic acids
  • Suitable for fresh or frozen plasma/serum from EDTA, citrate or Streck® Cell-free DNA BCT® tubes
  • Downstream compatibility with various assays - ddPCR, NGS, qPCR etc.
  • Compatibility with other automated Revvity systems

Application data

cfDNA was isolated from 1 ml, 2 ml, and 5 ml from two different donors both with the chemagic kits on the chemagic 360 instrument and manually with competitor. For cfDNA analysis, a short fragment (115 bp) from a consensus sequence with abundant genomic ALU repeats was amplified. The exemplary data from donor 2 shows that the yield of cfDNA is scalable to sample input.

Read more

Sprungziel Customer Review

Customer Review

Customer Review

“The chemagic™ cfDNA extraction kit on the chemagic 360 platform has greatly reduced the hands-on time needed for sample preparation. It has also given us improved cfDNA yields compared to the current, manual approach used in our laboratory and enabled significantly higher throughput. The ease-of-use of the system is high and has made it easy for us to rapidly train our staff and implement the approach in our day-to-day practice.”

Veli-Mikko Puupponen, CEO, BiopSense Oy/Ltd, Finland

“We use cfDNA extracted with chemagic™ technology in digital droplet PCR (ddPCR) assays to support diagnosis of colorectal cancer (Calleson et al., 2022), lung cancer (Frank et al., 2022) and anal cancer (based on HPV cfDNA detection, Lefèvre et al., 2021) with a sample to result turnaround time of 48 h.”

Professor Niels Pallisgaard, Dept of Pathology, Zealand University Hospital, Denmark

 

Webinar

Webinar

Webinar

The webinar focuses on optimization of the analytical steps including sampling, plasma isolation and purification (yield, degree of single stranded DNA, Proteinase K carryover and elution volume). Setup of ctDNA dPCR with dPCR assay controls, number of replicates and sample QC controls are discussed, including use of spike-in, measurement of contaminating lymphocyte DNA and DNA fragmentation. Strategies to select the optimal dPCR assay, identify germlines mutations, CHIPs and patients that are ctDNA non-shedders are presented. Finally, different ways to report ctDNA graphically are shown with pros and cons discussed.

Sprungziel Manual Option

 

Looking for a Manual Option?

If you do not have a chemagicTM instrument but would like to benefit from the advantage of chemagicTM M-PVA Magnetic Bead technology, the manual chemagicTM cfDNA 5k kit is now available. Process up to 5 ml of sample and avoid spillages and clogs linked to spin column or vacuum-based systems with an easy magnetic bead-based protocol. 

Manual cfDNA extraction with chemagic cfDNA 5k kit

Specifications:

  • 40 preps
  • 1-5 ml plasma/serum input
  • 30-100 µl elution volume
  • 60-90 minutes processing time

Additional magnetic racks required, see Product Overview for Manual DNA Isolation for details

Sprungziel Product Overview

Product overview for Automated cfDNA isolation

Catalog No.Kit NameInstrumentSamples/BatchPreps/KitSample VolumesProcessing Time
CMG-1444*chemagic
Prime™ cfDNA 5k Kit H24
chemagic Prime 4/8242403 - 5 ml240 min
CMG-1304*chemagic
cfDNA 5k Kit H24
chemagic 360/MSM I242403 - 5 ml100 min
(incl. 15 min hands-on time)
CMG-1302*chemagic
cfDNA 2k Kit H24
chemagic 360/MSM I242401 - 2 ml90 min
(incl. 15 min hands-on time)
CMG-1310*chemagic
cfDNA 10k Kit H24
chemagic 360/MSM I242406-10 ml115 min
(incl. 20 min hands-on time)
CMG-1318chemagic
cfDNA 18k Kit H12
chemagic 360/MSM I12Up to 25010, 15 or 18 ml125 min
(incl. 15 min hands-on time)
CMG-1396chemagic
cfDNA 1.5k Kit H96
chemagic 360/MSM I969600.5 - 1.5 ml120 min
(incl. 30 min hands-on time)

*Additional accessories required: 3 quantity chemagic 13 mL Double Bottom Rack H24 (art. No. CMG-13005340)

Product overview for Manual cfDNA isolation

Product No.Kit NameAccessories required
Preps / Kit
Sample Volumes Processing Time
CMG-134chemagic™ cfDNA 5k Kitchemagic™ Stand 2 x 12
chemagic Magnetic Stand Type F
chemagic cfDNA Stand 12 (Optional)
1 - 5 ml60-90 min (depending on user experience)

Sprungziel Manuals

Plasma Preparation Protocol

Best Practices in cfDNA Purification

Sample Collection & Plasma Preparation

To avoid contamination of cfDNA with genomic DNA from ruptured blood cells, it is recommended to prepare plasma as fresh as possible (within 24h after blood draw for EDTA tubes or longer for specialized cell free blood collection tubes, refer to tube manufacturer).
A double centrifugation protocol during plasma preparation is recommended to minimize the potential carry over of cells and genomic DNA (refer to tube manufacturer for max. centrifugation speeds). Special care should be taken to avoid transfer of any other blood components (buffy coat or red blood cells) when separating the plasma fraction.
From 10 ml of whole blood, approximately 4 - 5 ml plasma can be expected. For a guidance on how to prepare plasma for cfDNA analysis, please refer to manuals below.

cfDNA Quantification

cfDNA yields isolated from human plasma samples are typically in the range of 1 - 30 ng/mL of plasma and therefore critically low and maybe outside the detection parameters determined by spectrophotometric methods. If quantification of the extracted cfDNA is required, two methods are recommended: A PCR-based method (qPCR, ddPCR) and a fragment analysis assay where an internal reference is used such as Labchip cfDNA Assay. Quantification based on fluorometric method may lead to varying results dependent on various factors and therefore is not recommended. Likewise, fragment analysis methods based on upper marker peak size will lead to inaccurate quantitation and should be avoided. Please refer to manuals for more details.

Download Manual

Catalog No.Kit NamePDF Protocol
CMG-134chemagic™ cfDNA 5k Kit (manual)Protocol CMG-134 chemagic cfDNA 5k Kit
CMG-1304chemagic™ cfDNA 5k Kit H24Protocol CMG-1304 chemagic cfDNA 5k Kit H24
CMG-1302chemagic™ cfDNA 2k Kit H24Protocol CMG-1302 chemagic cfDNA 2k Kit H24
CMG-1396chemagic™ cfDNA 1.5k Kit H96Protocol CMG-1396 chemagic cfDNA 1.5k Kit H96
CMG-1310chemagic™ cfDNA 10k Kit H24Protocol CMG-1310 chemagic cfDNA 10k Kit H24

Sprungziel Application Notes

Application Notes

Contact us for further information










    References

    Kwon, HJ., Shin, S.H., Kim, H.H. et al. Advances in methylation analysis of liquid biopsy in early cancer detection of colorectal and lung cancer. Sci Rep 13, 13502 (2023).

    Frank, M. S., Andersen, C. S. A., Ahlborn, L. B., Pallisgaard, N., Bodtger, U., & Gehl, J. Circulating Tumor DNA Monitoring Reveals Molecular Progression before Radiologic Progression in a Real-life Cohort of Patients with Advanced Non–small Cell Lung Cancer. Cancer Research Communications, 2022; 2(10), 1174–1187.

    Callesen LB, Hansen TF, Andersen RF, Pallisgaard N, Kramer S, Schlander S, Rafaelsen SR, Boysen AK, Jensen LH, Jakobsen A, Spindler KG. OPTIMISE: Optimisation of treatment selection and follow-up in oligometastatic colorectal cancer - a ctDNA-guided phase II randomised approach. Study protocol. Acta Oncol. 2022 Sep;61(9):1152-1156.

    Kirchweger P, Kupferthaler A, Burghofer J, Webersinke G, Jukic E, Schwendinger S, Wundsam H, Biebl M, Petzer A, Rumpold H. Prediction of response to systemic treatment by kinetics of circulating tumor DNA in metastatic pancreatic cancer. Front Oncol. 2022 Aug 30;12:902177. 

    Kirchweger P, Kupferthaler A, Burghofer J, Webersinke G, Jukic E, Schwendinger S, Weitzendorfer M, Petzer A, Függer R, Rumpold H, Wundsam H. Circulating tumor DNA correlates with tumor burden and predicts outcome in pancreatic cancer irrespective of tumor stage. Eur J Surg Oncol. 2022 May;48(5):1046-1053.

    Broholm M, Bojesen R, Gögenur M, Weinberger Rosen A, Watt S, Bulut M, Vogelsang R, Quist Jensen H, Orhan A, Bjerrum E, Søs Auður Andersen C, Pallisgaard N, Litman T, Troelsen JT, Gögenur I. Circulating Cell-Free DNA and Systemic Inflammatory Response after Self-Expandable Metal Stent for Malignant Bowel Obstruction. Archives of Microbiology and Immunology 6 (2022): 247-255.

    Lefèvre AC, Pallisgaard N, Kronborg C, Wind KL, Krag SRP, Spindler KG. The Clinical Value of Measuring Circulating HPV DNA during Chemo-Radiotherapy in Squamous Cell Carcinoma of the Anus. Cancers (Basel). 2021 May 18;13(10):2451.

    Yu J, Cho E, Choi J, Lim JE, Lee J, Kang M, Sung HH, Jeong BC, Seo SI, Jeon SS, Lee HM, Jeon HG. Genomic mutation profiling using liquid biopsy in Korean patients with prostate cancer: Circulating tumor DNA mutation predicts the development of castration resistance. Investig Clin Urol. 2021 Mar;62(2):224-232.

    Moon SM, Kim JH, Kim SK, Kim S, Kwon HJ, Bae JS, Lee S, Lee HS, Choi MY, Jeon BH, Jeong BH, Lee K, Kim HK, Kim J, Um SW. Clinical Utility of Combined Circulating Tumor Cell and Circulating Tumor DNA Assays for Diagnosis of Primary Lung Cancer. Anticancer Res. 2020 Jun;40(6):3435-3444.

    For research use only. Not for use in diagnostic procedures.