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
Center for Oncological Research Antwerp (CORE), University of Antwerp (UAntwerp), 2610 Wilrijk, Belgium.
Laboratory of Pathological Anatomy, Antwerp University Hospital (UZA), 2650 Edegem, Belgium.
Cancers (Basel). 2019 Mar 30;11(4):458. doi: 10.3390/cancers11040458.
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 K₂EDTA (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 -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.
循环游离（肿瘤）DNA（cfDNA/ctDNA）和循环游离（肿瘤）RNA（cfRNA/ctRNA）的联合分析在确定癌症患者的分子特征方面显示出巨大潜力。优化工作流程对于获得一致且可重复的结果是必要的。在本研究中，我们比较了五种离心方案，以实现cfDNA/ctDNA和cfRNA/ctRNA的最佳产量。这些方案在离心速度、环境温度、时间和离心步骤数量方面有所不同。从33名参与者中采集的样本收集于BD Vacutainer K₂EDTA（EDTA）管或游离DNA BCT（Streck）管中。通过数字液滴PCR（ddPCR）和定量PCR（qPCR）对所有样本中的cfDNA浓度和片段大小以及cfRNA浓度进行定量。通过ddPCR确定突变的ctDNA和ctRNA分数。在EDTA管中，发现同时产生血浆和血小板的方案可产生高质量的cfDNA和cfRNA浓度。两步高速离心方案与高cfDNA浓度但低cfRNA浓度相关。一步低速方案产生高cfRNA浓度。然而，这同时伴随着大量基因组DNA（gDNA）污染。在Streck管中，两步高速离心方案也产生了高质量、高cfDNA浓度。然而，这些管与cfRNA分析不兼容。
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