Influence of plasma processing on recovery and analysis of circulating nucleic acids.
Influence of plasma processing on recovery and analysis of circulating nucleic acids.
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DOI：
10.1371/journal.pone.0077963
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Shaw JA
中科院分区：
文献类型：
--
作者：
Page K;Guttery DS;Zahra N;Primrose L;Elshaw SR;Pringle JH;Blighe K;Marchese SD;Hills A;Woodley L;Stebbing J;Coombes RC;Shaw JA
循环核酸 (CNA) 正在作为癌症液体活检进行研究。然而，血液处理以及循环游离 DNA (cfDNA) 和 microRNA (miRNA) 的分离方法存在很大差异。在这里，我们使用掺入参考模板来比较 4 种市售 cfDNA 试剂盒和 3 种 miRNA 试剂盒的提取效率和重现性。我们还比较了增加静脉穿刺和离心之间的时间以及差速离心力对 CNA 恢复的影响。通过 TaqMan qPCR 和靶向深度测序对 cfDNA 进行定量。使用 TaqMan 低密度阵列和测定法评估 miRNA 谱。 QIAamp® DNA Blood Mini 和循环核酸试剂盒可实现最高的 cfDNA 回收率和 564bp 掺入的有效回收率 (>90%)。此外，靶向测序揭示了重叠的 cfDNA 图谱和变异深度，包括使用 Ion AmpliSeq™Cancer Hotspot Panel v2 检测 HER2 基因扩增。使用 miRNeasy 血清/血浆试剂盒获得了最高产量的 miRNA 和合成拟南芥 miR-159a 掺入物，血浆饱和度高于 200 µl。随着离心前时间的增加 (p<0.001) 和离心力的增加，随着血小板相关 miRNA 的耗尽，miRNA 谱显示出显着变化，而 cfDNA 不受影响。然而，样品重复在 TaqMan 低密度阵列上显示出出色的重现性（ρ = 0.96，p<0.0001）。我们还成功地为储存超过 12 年的血浆样本生成了 miRNA 图谱，凸显了分析储存样本生物库的潜力。在液体活检时代，需要方法标准化以尽量减少变异，尤其是 miRNA。
Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.
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影响因子：
14.9
作者：
Kozomara A;Griffiths-Jones S
通讯作者：
Griffiths-Jones S
影响因子：
3.7
作者：
Cheng HR;Liu CJ;Tseng TC;Su TH;Yang HI;Chen CJ;Kao JH
通讯作者：
Kao JH
影响因子：
2.2
作者：
Shaw, Jacqui A.;Brown, James;Stebbing, Justin
通讯作者：
Stebbing, Justin
DOI：
10.1093/jnci/dji432
发表时间：
2005-12-21
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
Sozzi, G;Roz, L;Pastorino, U
通讯作者：
Pastorino, U
影响因子：
3.7
作者：
Duttagupta R;Jiang R;Gollub J;Getts RC;Jones KW
通讯作者：
Jones KW