Microfluidic mRNA Integrity Assay

描述（由申请人提供）：人类基因组测序开始对特定基因在人类疾病中的作用产生深刻的见解。无论是通过使用qPCR、微阵列还是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、确定药物开发靶点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容易降解，从而影响测量到的mRNA水平，因此研究人员通常在将RNA样品用于下游分析（如qPCR、微阵列或RNAseq）之前，先测试样品的完整性。广泛使用的基于总RNA电泳图预测mRNA完整性的方法是不完美的预测下游分析样本的充分性的方法。这导致在定量基因表达水平方面存在更大的可变性，并增加了在昂贵的测定中运行降解样品的成本，或浪费了被错误地判断为已降解的优质样品。我们建议开发一种改进的mRNA完整性分析，产生类似于Northern blot的结果，但格式更方便。提议的分析将使用荧光标记探针和微电泳来监测特定mRNA物种的降解。通过观察mRNA而不是总RNA，并使用直接的电泳分析，新的分析方法有望更好地预测RNA样品对mRNA分析的适用性，特别是对FFPE样品。当使用qPCR、微阵列或RNAseq测量mRNA水平时，这种分析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.