Implementation of innovative RNA sample quality control methods

描述(由申请人提供)：包括转录丰度(TA)测量在内的分子诊断测试具有更好地预测癌症风险、预后和每个人的最佳治疗方案的巨大潜力。然而，这一潜力受到临床样本收集和处理方法的现状的限制。今天通常采用的方法产生的临床样本在RNA质量上差异很大。改进这些方法的努力在一定程度上受到了测量RNA质量手段不足的限制。此外，由于不充分的RNA质量控制(QC)测量方法，现有的具有可变RNA质量的生物谱库的使用受到限制。这阻碍了建立适当的截止标准，以确定哪些样本将产生可靠的逆转录聚合酶链式反应(RT-PCR)结果。RNA质量的样本间差异的来源包括RNA完整性、基因组DNA(GDNA)污染，以及a)干扰RT效率和/或b)携带到cDNA并导致基因特异性的PCR效率抑制的物质或方法。在先导性研究中，对控制RNA降解的ACTB cDNA分子/ng RNA进行了量化，对控制gDNA污染的RNA中的cc10基因gDNA进行了量化，并测量了每个基因的转录丰度与控制聚合酶链式反应抑制剂的相应内标(IS)分子的已知数量的关系。在这个拟议的项目中，将开展更全面的调查，以寻找最佳的RNA完整性测试，并在新型标准化纳米阵列聚合酶链式反应(SNAP)平台上实施gDNA污染和RT抑制测试。然后，这些方法将被应用于指导改进的生物样本收集方法的开发。具体目标是：目的1.建立可靠的测试来测量RNA完整性、gDNA污染和RT抑制以及伴随的参考物质；目的2.使用稳健的RNA质量控制方法来建立可靠的阈值，用于RT-PCR或微阵列基于转录丰度的诊断测试，并指导临床生物检材收集和处理方法的改进。目的3.论证在商业平台上单独或与疾病特异性检测一起实施RNA质控检测的可行性。拟议项目的成功完成将有助于确定更有可能产生适合于分子诊断研究的RNA样本的组织样本收集和处理方法。此外，它将能够更准确地确定根据目前的方法和/或在现有组织库中收集的哪些临床生物标本将为基于转录本丰度的诊断测试产生可靠的结果。 公共卫生相关性：人类基因组测序为基于转录丰度测量的诊断进展打开了机会。由于缺乏足够的RNA质量测试，这一承诺受到了阻碍。改进的RNA质量控制测试的开发将使改进的生物样品收集方法和改进的方法得以开发，以确定哪些RNA样本具有足够的质量来产生可靠的转录丰度值。这些进展将导致癌症诊断和管理领域中个性化药物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.