Collection, Stabilization and Storage of Saliva Samples for Cancer Research

描述（由申请人提供）：在早期阶段诊断癌症的能力将大大提高治疗成功的机会，并降低死亡率和发病率。目前还没有分子检测可以非侵入性地诊断或筛查口腔癌。我们能够确定一个7基因签名，可以预测口腔癌的存在，准确率为82%。自2004年以来，我们的实验室一直在积极追求人类唾液RNA的表征和诊断用途。 我们的提案“癌症研究唾液样本的收集、稳定和储存”是对RFA题为“癌症样本制备创新”（RFA CA-07-037）的回应。我们将开发最佳和标准化的收集，RNA稳定，储存和处理方案，以进行唾液mRNA的基因表达分析。我们提出了三个具体目标，以解决这一RFA的多方面性质。目的1建立唾液中mRNA的最佳提取方法。在目标2中，我们将确定mRNA水平的个体内波动，定义足够的内源性转录物用于标准化，并在独立队列中验证我们的口腔癌标志物和其他新候选物。在目标3中，我们将确定唾液和表达模式在不同储存条件和稳定试剂下长达6个月的稳定性。在所有的目标，我们将应用一种新的方法，多重逆转录和预扩增与随后的定量PCR的各种mRNA转录内源性唾液。此外，我们的设置允许实现几个尖峰序列，这将产生重要的见解，在提取和存储，是临床应用的理想控制。我们有信心，我们的研究可以直接转化为一项研究，用于口腔癌标志物的大规模验证，并引发了众多项目和样本收集的启动，以使用唾液作为口腔和全身性疾病的诊断工具。我们的研究将有助于人类唾液作为临床上重要的体液进行分子诊断，以改善癌症导向的医疗保健。

DESCRIPTION (provided by applicant): The ability to diagnose cancer at an early stage will greatly enhance chances of treatment success and reduce the mortality and morbidity. Currently there is no molecular test that can diagnose or screen for oral cancer non-invasively. We were able to identify a 7-gene signature that can predict the presence of an oral cancer with an accuracy of 82%. Since 2004, the characterization and diagnostic use of human salivary RNA has been actively pursued in our laboratory. Our proposal, "Collection, Stabilization and Storage of Saliva Samples for Cancer Research," responds to the RFA titled "Innovations in Cancer Sample Preparation" (RFA CA-07-037). We will develop optimal and standardized collection, RNA stabilization, storage and processing protocols to perform gene expression analysis of salivary mRNA. We propose three Specific Aims to address the multifaceted nature of this RFA. Aim 1 will establish optimal extraction methods for mRNA from saliva. In Aim 2 we will determine the intra-individual fluctuation of mRNA levels, define adequate endogenous transcripts for normalization and validate our oral cancer markers and additional new candidates in an independent cohort. In Aim 3 we will determine stability of saliva and expression patterns over periods of up to 6 months with different storage conditions and with stabilizing reagent. In all aims we will apply a new method for the multiplex reverse transcription and pre-amplification with subsequent quantitative PCR of a variety of mRNA transcripts endogenous to saliva. In addition, our set-up allows the implementation of several spike sequences that will yield important insights in the extraction and storage and are ideal controls for clinical application. We are confident that our research can be translated directly into a study for the large validation of oral cancer markers and spark the initiation of a multitude of projects and sample collections for the use of saliva as a diagnostic tool for oral and systemic diseases. Our studies will contribute to the advancement of human saliva as a clinically important body fluid for molecular diagnosis to improve cancer-directed health care.