Rice Jonathan, Roberts Henry, Burton James, Pan Jianmin, States Vanessa, Rai Shesh N, Galandiuk Susan
Price Institute of Surgical Research, Hiram C. Polk Jr., M.D. Department of Surgery, University of Louisville School of Medicine, Louisville, KY, United States of America.
Department of Bioinformatics and Biostatistics, University of Louisville School of Public Health and Information Sciences, Louisville, KY, United States of America.
PLoS One. 2015 Apr 8;10(4):e0121948. doi: 10.1371/journal.pone.0121948. eCollection 2015.
There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct) setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas) and 16 patients without any neoplasm (controls). Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy). We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4%) documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ≤12 h provided consistent ΔCt. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively). A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy. For standardized reporting, we recommend plasma extraction ≤ 12 h, using modified miRNeasy extraction and utilizing a 0.03 Ct.
血浆微小RNA（miRNA）作为人类疾病生物标志物的报道日益增多，但方法学报告的标准却很少，导致数据不一致。我们系统回顾了2013年7月至2014年6月发表的血浆miRNA研究，以评估其方法学。研究了六个参数：血浆提取时间、RNA提取方法、miRNA类型、定量、循环阈值（Ct）设置以及统计分析方法。我们将这些数据与一种提议的标准方法学技术进行了比较。首先使用微流控芯片技术对380种miRNA进行初步筛选，并在另一组患者中进行验证，我们比较了16例良性结直肠肿瘤（高级别腺瘤）患者和16例无任何肿瘤患者（对照组）之间差异表达的11种miRNA。在采血后立即、12、24、48或72小时分离血浆。使用两种不同技术（预扩增的Trizol LS或改良的miRNeasy）提取miRNA。我们对11种miRNA进行了基于Taqman的逆转录-聚合酶链反应（RT-PCR）检测，随后使用可变Ct设置或固定Ct值（0.01、0.03、0.05或0.5）进行分析。检测由两名不同的操作人员重复进行。RNU6作为内参。系统回顾产生了74篇符合纳入标准的手稿。一篇手稿（1.4%）记录了所有6个方法学参数，而<5%的研究列出了Ct设置。在我们提议的标准技术中，血浆提取≤12小时可提供一致的ΔCt。与Trizol LS相比，miRNeasy提取产生更高的miRNA浓度和更少未表达的miRNA（分别为1/704种miRNA [0.14%]和109/704种miRNA [15%]未表达）。固定Ct值设置为0.03可产生最具可重复性的数据，前提是<10%的miRNA未表达。操作人员内部无显著差异。使用Trizol LS提取时操作人员之间存在显著差异，而使用改良的miRNeasy时这种差异可忽略不计。为了标准化报告，我们建议血浆提取≤12小时，使用改良的miRNeasy提取并采用0.03的Ct值。
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