University of Otago, Department of Medicine, P.O. Box 56, Dunedin, 9054, New Zealand.
University of Otago Christchurch, Department of Medicine, P.O. Box 4345, Christchurch, 8140, New Zealand.
Sci Rep. 2017 Dec 8;7(1):17235. doi: 10.1038/s41598-017-17619-0.
Subtle molecular differences indicate the heterogeneity present in a number of disease settings. Digital-PCR (dPCR) platforms achieve the necessary levels of sensitivity and accuracy over standard quantitative RT-PCR (qPCR) that promote their use for such situations, detecting low abundance transcript and subtle changes from gene expression. An underlying requisite is good quality RNA, principally dictated by appropriate tissue handling and RNA extraction. Here we consider the application of dPCR to measures of gene expression in pathological tissues with inherent necrosis, focusing on rheumatoid subcutaneous nodules. Variable RNA fragmentation is a feature of RNA from such tissues. Increased presence of transcript fragmentation is reflected in a proportionate decrease in Agilent DV metric and downstream, a reduction in endogenous control genes' expression, measured by RT-dPCR. We show that normalisation of target gene expression to that for endogenous control genes sufficiently corrects for the variable level of fragmented RNA. Recovery of target gene values was achieved in samples comprising as much as 50 percent fragmented RNA, indicating the suitability and appropriate limitation of such data treatment when applied to samples obtained from inherently necrotic tissues.
分子细微差异表明，在许多疾病环境中存在异质性。数字 PCR (dPCR) 平台在标准定量 RT-PCR (qPCR) 基础上实现了必要的灵敏度和准确性，促进了其在这些情况下的应用，可检测低丰度转录本和基因表达的细微变化。一个基本要求是高质量的 RNA，主要由适当的组织处理和 RNA 提取决定。在这里，我们考虑将 dPCR 应用于存在固有坏死的病理组织中的基因表达测量，重点是类风湿性皮下结节。来自此类组织的 RNA 存在可变的 RNA 片段化是其特征。转录本片段化程度增加反映在 Agilent DV 指标相应下降，下游内源性对照基因的表达也随之减少，通过 RT-dPCR 进行测量。我们表明，将目标基因表达与内源性对照基因的表达进行归一化可以充分纠正 RNA 片段化的差异。在包含高达 50%片段化 RNA 的样本中可以回收目标基因值，这表明当应用于从固有坏死组织获得的样本时，这种数据处理方法具有适用性和适当的局限性。
