Implementation of innovative RNA sample quality control methods

描述（由申请人提供）：包括转录本丰度（TA）测量的分子诊断测试具有更好地预测癌症风险、预后和每个个体的最佳治疗的巨大潜力。然而，这种潜力受到临床样本采集和处理方法现状的限制。目前通常采用的方法产生的临床样品在RNA质量方面存在很大差异。改进这些方法的努力部分受到测量RNA质量的手段不足的限制。此外，由于RNA质量控制（QC）测量方法不足，限制了具有可变RNA质量的现有生物样本库的使用。这妨碍了建立适当的截止标准，以确定哪些样品将产生可靠的逆转录酶聚合酶链反应（RT-PCR）结果。RNA质量的样本间变异来源包括RNA完整性、基因组DNA（gDNA）污染以及a）干扰RT效率和/或B）携带至cDNA并导致基因特异性抑制PCR效率的物质或方法。在初步研究中，定量ACTB cDNA分子/ng RNA控制RNA降解的范围比电泳图测量的范围更广，定量RNA中CC 10基因gDNA控制gDNA污染，并测量每个基因相对于已知数量的相应内标（IS）分子的转录本丰度控制PCR抑制剂。在该拟议项目中，将开发更全面的研究，以获得RNA完整性的最佳检测方法，并将沿着在新型标准化NanoArray PCR（SNAP）平台上进行gDNA污染和RT抑制检测。然后，这些方法将被应用于指导生物标本采集的改进方法的开发。具体目标是：目标1。建立稳健检测，以测量RNA完整性、gDNA污染和RT抑制沿着以及随附参比物质目标2。使用强大的RNA QC方法建立截断阈值，用于通过RT-PCR或微阵列可靠测量基于转录本丰度的诊断测试，并指导临床生物标本采集和处理方法的改进。目标3.证明在商业平台上单独或与疾病特异性检测一起实施RNA QC检测的可行性。成功完成拟议项目将能够确定更有可能产生适合分子诊断研究的RNA样本的组织样本收集和处理方法。此外，它将使得能够更准确地确定根据本发明的方法和/或在现有的组织库中收集的哪些临床生物样本将产生可靠的结果，用于有希望的基于转录本丰度的诊断测试。 公共卫生相关性：人类基因组的测序为基于转录本丰度测量的诊断学的进步打开了机会。由于缺乏足够的RNA质量测试，这一前景受到了阻碍。改进的RNA质量控制测试的开发将能够开发改进的生物样本收集方法和改进的方法，以确定哪些RNA样品具有足够的质量来产生可靠的转录本丰度值。这些进展将导致癌症诊断和管理领域个性化医疗所需的更可靠的测试。

DESCRIPTION (provided by applicant): Molecular diagnostic tests comprising transcript abundance (TA) measurement have great potential to better predict cancer risk, prognosis, and the optimal therapeutic for each individual. However, this potential is limited by the status of clinical sample collection and processing methods. Methods typically practiced today yield clinical samples with wide variation in RNA quality. Efforts to improve these methods are limited in part by inadequate means to measure RNA quality. Further, use of existing banks of biospecimens with variable RNA quality is limited due to inadequate RNA quality control (QC) measurement methods. This prevents establishment of appropriate cut-off criteria by which to determine which samples will yield reliable reverse transcriptase polymerase chain reaction (RT-PCR) results. Sources of inter-sample variation in RNA quality include RNA integrity, genomic DNA (gDNA) contamination, and substances or methods that either a) interfere with RT efficiency, and/or b) carry over to cDNA and cause gene-specific inhibition of PCR efficiency. In pilot studies, quantification of ACTB cDNA molecules/ng RNA controlled for RNA degradation over wider scale than that measured by electropherogram, quantification of CC10 gene gDNA in RNA controlled for gDNA contamination, and measurement of transcript abundance of each gene relative to known number of respective internal standard (IS) molecules controlled for PCR inhibitors. In this proposed project, more comprehensive investigation for the best tests for RNA integrity will be developed and these will be implemented, along with the tests for gDNA contamination and RT inhibition on the novel Standardized NanoArray PCR (SNAP) platform. These methods then will be applied to guide development of improved methods for biospecimen collection. The specific aims are: Aim 1. Establish robust tests to measure RNA integrity, gDNA contamination, and RT inhibition along with accompanying reference materials Aim 2. Use the robust RNA QC methods to establish cut-off thresholds for reliable measurement of transcript abundance based diagnostic tests by RT-PCR or microarray and guide improvements in methods for collecting and processing clinical biospecimens. Aim 3. Demonstrate the feasibility of implementing the RNA QC tests either alone or together with disease specific assays on commercial platforms. Successful completion of the proposed project will enable identification of tissue sample collection and processing methods that are more likely to yield RNA samples suitable for molecular diagnostic studies. Further, it will enable more accurate determination of which clinical biospecimens collected according to present methods and/or in existing tissue banks will yield reliable results for promising transcript abundance based diagnostic tests. PUBLIC HEALTH RELEVANCE: Sequencing of the human genome has opened opportunities for advances in diagnostics based on transcript abundance measurement. This promise has been hampered by lack of adequate tests for RNA quality. Development of improved RNA Quality Control tests will enable development of improved biospecimen collection methods and improved methods to determine which RNA samples have sufficient quality to yield reliable transcript abundance values. These advances will lead to more reliable testing required for personalized medicine in the field of cancer diagnosis and management.