Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol

Laure Sorber, Karen Zwaenepoel, Julie Jacobs, Koen de Winne, Sofie Goethals, Pablo Reclusa, Kaat van Casteren, Elien Augustus, Filip Lardon, Geert Roeyen, Marc Peeters, Jan van Meerbeeck, Christian Rolfo, Patrick Pauwels

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K 2 EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.
Original languageEnglish
Article number458
JournalCancers
Volume11
Issue number4
DOIs
Publication statusPublished - 2019
Externally publishedYes

Cite this

Sorber, L., Zwaenepoel, K., Jacobs, J., de Winne, K., Goethals, S., Reclusa, P., ... Pauwels, P. (2019). Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol. Cancers, 11(4), [458]. https://doi.org/10.3390/cancers11040458
Sorber, Laure ; Zwaenepoel, Karen ; Jacobs, Julie ; de Winne, Koen ; Goethals, Sofie ; Reclusa, Pablo ; van Casteren, Kaat ; Augustus, Elien ; Lardon, Filip ; Roeyen, Geert ; Peeters, Marc ; van Meerbeeck, Jan ; Rolfo, Christian ; Pauwels, Patrick. / Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol. In: Cancers. 2019 ; Vol. 11, No. 4.
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abstract = "The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K 2 EDTA (EDTA) tubes or cell-free DNA BCT{\circledR} (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.",
author = "Laure Sorber and Karen Zwaenepoel and Julie Jacobs and {de Winne}, Koen and Sofie Goethals and Pablo Reclusa and {van Casteren}, Kaat and Elien Augustus and Filip Lardon and Geert Roeyen and Marc Peeters and {van Meerbeeck}, Jan and Christian Rolfo and Patrick Pauwels",
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Sorber, L, Zwaenepoel, K, Jacobs, J, de Winne, K, Goethals, S, Reclusa, P, van Casteren, K, Augustus, E, Lardon, F, Roeyen, G, Peeters, M, van Meerbeeck, J, Rolfo, C & Pauwels, P 2019, 'Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol' Cancers, vol. 11, no. 4, 458. https://doi.org/10.3390/cancers11040458

Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol. / Sorber, Laure; Zwaenepoel, Karen; Jacobs, Julie; de Winne, Koen; Goethals, Sofie; Reclusa, Pablo; van Casteren, Kaat; Augustus, Elien; Lardon, Filip; Roeyen, Geert; Peeters, Marc; van Meerbeeck, Jan; Rolfo, Christian; Pauwels, Patrick.

In: Cancers, Vol. 11, No. 4, 458, 2019.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol

AU - Sorber, Laure

AU - Zwaenepoel, Karen

AU - Jacobs, Julie

AU - de Winne, Koen

AU - Goethals, Sofie

AU - Reclusa, Pablo

AU - van Casteren, Kaat

AU - Augustus, Elien

AU - Lardon, Filip

AU - Roeyen, Geert

AU - Peeters, Marc

AU - van Meerbeeck, Jan

AU - Rolfo, Christian

AU - Pauwels, Patrick

PY - 2019

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N2 - The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K 2 EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

AB - The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K 2 EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

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UR - https://www.ncbi.nlm.nih.gov/pubmed/30935089

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Sorber L, Zwaenepoel K, Jacobs J, de Winne K, Goethals S, Reclusa P et al. Circulating cell-free DNA and RNA analysis as liquid biopsy: Optimal centrifugation protocol. Cancers. 2019;11(4). 458. https://doi.org/10.3390/cancers11040458