Evaluation of pre-analytical factors affecting plasma DNA analysis.
Evaluation of pre-analytical factors affecting plasma DNA analysis.
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DOI：
10.1038/s41598-018-25810-0
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发表时间：
2018-05-09
影响因子：
4.6
通讯作者：
Murtaza M
中科院分区：
文献类型：
--
作者：
Markus H;Contente-Cuomo T;Farooq M;Liang WS;Borad MJ;Sivakumar S;Gollins S;Tran NL;Dhruv HD;Berens ME;Bryce A;Sekulic A;Ribas A;Trent JM;LoRusso PM;Murtaza M
分析前因素会显著影响循环无细胞DNA(CfDNA)分析。然而，几乎没有可靠的方法来快速评估样品质量和分析前处理的影响。为了弥补这一空白，并评价DNA提取方法和采血管对cfDNA产量和片段大小的影响，我们建立了针对单拷贝基因组座位的5个短和4个长扩增片段的多重滴状数字聚合酶链式反应(DdPCR)。用该方法对7种cfDNA提取试剂盒进行了比较，发现cfDNA的得率和片段大小差异显著。我们还用23名健康志愿者的血浆样本比较了3种采血方案(EDTA管在1 小时内处理，无细胞DNA采血管在24和72 小时内处理)，发现这些方案在cfDNA产量、片段大小和背景噪声方面没有显著差异。在219例临床样本中，静脉穿刺术后立即处理的血浆样本的cfDNA片段比存档样本短，提示背景DNA来自裂解的外周血细胞。综上所述，我们描述了一种在下游分子分析之前评估cfDNA样本质量的多重ddPCR分析方法，并评估了cfDNA研究中分析前变异的潜在来源。
Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.
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影响因子：
17.1
作者：
De Vlaminck I;Valantine HA;Snyder TM;Strehl C;Cohen G;Luikart H;Neff NF;Okamoto J;Bernstein D;Weisshaar D;Quake SR;Khush KK
通讯作者：
Khush KK
DOI：
10.1073/pnas.1500076112
发表时间：
2015-03-17
影响因子：
11.1
作者：
Jiang, Peiyong;Chan, Carol W. M.;Lo, Y. M. Dennis
通讯作者：
Lo, Y. M. Dennis
影响因子：
5
作者：
Fleischhacker, Michael;Schmidt, Bernd;Holdenrieder, Stefan
通讯作者：
Holdenrieder, Stefan
影响因子：
158.5
作者：
Bianchi, Diana W.;Parker, R. Lamar;Sehnert, Amy J.
通讯作者：
Sehnert, Amy J.
影响因子：
4.3
作者：
Devonshire, Alison S.;Whale, Alexandra S.;Gutteridge, Alice;Jones, Gerwyn;Cowen, Simon;Foy, Carole A.;Huggett, Jim F.
通讯作者：
Huggett, Jim F.