Microfluidic mRNA Integrity Assay

微流控 mRNA 完整性检测

• 批准号: 8633052
• 负责人: Javier Farinas
• 金额: $ 13.49万
• 依托单位: CAERUS MOLECULAR DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-07 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8633052
• 关键词: Actins; Affect; Algorithms; Biological; Biological Assay; Biological Neural Networks; Buffers; Chemicals; Clinical; Cost Analysis; Data Analyses; Detection; Diagnostic; Disease; Electrophoresis; Ensure; Failure; Formalin; Gel; Gene Expression; Gene Expression Profiling; Genes; Goals; Housekeeping Gene; Human Genome; Label; Lead; Length; Measurement; Measures; Messenger RNA; Methods; Microfluidics; Molecular; Monitor; Northern Blotting; Paraffin Embedding; Patient Care; Process; RNA; RNA Degradation; Research Personnel; Ribosomal RNA; Role; Running; Sampling; Series; Testing; Transcript; base; clinical decision-making; cost; design; drug development; gene function; human disease; improved; insight; mRNA Transcript Degradation; nanoassay; public health relevance; therapeutic target; wasting

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.

描述（由申请人提供）：人类基因组测序开始对人类疾病中特定基因的作用产生深刻的认识。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、确定药物开发的靶点以及越来越多地为患者护理做出临床决策都至关重要。由于RNA会发生降解，从而影响测量的mRNA水平，因此研究人员通常会在将样本用于下游检测（如qPCR、微阵列或RNAseq）之前测试RNA样本的完整性。基于总RNA电泳图预测mRNA完整性的广泛使用的方法是下游测定样品充分性的不完善预测。这导致在定量基因表达水平方面的更大的可变性和与在昂贵的测定中运行降解的样品或浪费被错误地判断为降解的良好样品相关的增加的成本。我们建议开发一种改进的mRNA完整性检测，产生类似于北方印迹的结果，但在一个方便的格式。拟议的测定将使用荧光标记探针和微电泳来监测特定mRNA种类的降解。通过观察mRNA而不是总RNA，并使用直接的电泳图分析，新的测定法有望更好地预测RNA样品对mRNA分析的适用性，特别是对于FFPE样品。当使用qPCR、微阵列或RNAseq测量mRNA水平时，预计这种测定法会给出更准确的结果。