Microfluidic mRNA Integrity Assay

描述(申请人提供)：人类基因组的测序开始对特定基因在人类疾病中的作用产生巨大的洞察力。无论是通过使用qPCR、微阵列还是通过RNAseq，监测基因表达水平的能力对于确定特定基因在疾病状态中的作用、确定药物开发的目标以及越来越多地为患者护理做出临床决策至关重要。由于RNA会被降解，这可能会影响测量的mRNA水平，因此研究人员通常会在将RNA样本用于下游分析(如qPCR、微阵列或RNAseq)之前测试该样本的完整性。广泛使用的根据总RNA的电泳图预测信使核糖核酸完整性的方法不能很好地预测样本是否适合进行下游分析。这导致了量化基因表达水平的更大变异性，并增加了与在昂贵的分析中运行降解的样本或浪费被错误判断为降解的好样本相关的成本。我们建议开发一种改进的mRNA完整性分析，其结果类似于Northern印迹，但以一种方便的形式。建议的分析将使用荧光标记的探针和微电泳法来监测特定mRNA物种的降解。通过观察mRNA而不是总RNA并使用直接的电泳图分析，新的分析方法有望更好地预测RNA样本是否适合进行mRNA分析，特别是FFPE样本。当使用定量聚合酶链式反应、微阵列或RNAseq来测量信使核糖核酸水平时，这种分析方法有望给出更准确的结果。

DESCRIPTION (provided by applicant): The sequencing of the human genome is beginning to yield great insight into the role of specific genes in human diseases. Whether through the use of qPCR, microarrays or by RNAseq, the ability to monitor gene expression levels is critical to identifying the role of specific genes in disease states, identifying targets for drug development and increasingly to make clinical decisions for patient care. Because RNA is subject to degradation that can affect measured mRNA levels, researchers typically test the integrity of an RNA sample before using the sample in a downstream assay such as qPCR, microarrays or RNAseq. Widely used methods which predict mRNA integrity based on electropherograms of total RNA are imperfect predictors of the adequacy of a sample for downstream assays. This leads to greater variability in quantifying gene expression levels and increased costs associated with running degraded samples in expensive assays or wasting of good samples which are wrongly judged to be degraded. We propose to develop an improved mRNA integrity assay that yields results similar to a Northern blot but in a convenient format. The proposed assay would use fluorescently labeled probes and microelectrophoresis to monitor the degradation of a specific mRNA species. By looking at mRNA rather than total RNA and using straightforward electropherogram analysis, the new assay is expected to be a better predictor of the suitability of an RNA sample for mRNA analysis particularly for FFPE samples. Such an assay is expected to give more accurate results when mRNA levels are measured using qPCR, microarrays or RNAseq.