Nanobind FFPE DNA/RNA Extraction

描述(由申请人提供)：Nanobind FFPE DNA/RNA提取尽管分子生物标记物的需求日益增长，并显示出潜在的优势，但事实证明，在患者的诊断和管理中常规使用它们是困难的。这一失败的原因之一是在临床环境中获取、快速处理、储存和运输快速冷冻组织样本的后勤挑战。标准的医院组织处理包括在甲醛中固定，然后包埋在石蜡块中，以产生福尔马林固定的石蜡包埋(FFPE)组织样本。如果这些FFPE样本能够被用于常规的分子分析，那么就有可能彻底改变目前的医疗实践。然后，在医院病理科获得的FFPE块可以用新的分子方法进行常规检测。此外，由于FFPE样本通常由医院病理科储存多年，也可以进行回溯性分子评估，使研究人员能够对具有已知临床结果的大型队列进行分子流行病学研究。我们的目标是开发一种新的DNA/RNA FFPE提取方法，该方法基于一种新的廉价制造的纳米材料-纳米结合。Nanobind是一种热塑性衬底，包含微米级褶皱和纳米级二氧化硅薄片的分层地形。与赋予DNA/RNA碎裂剪切力的珠子和柱子不同，无孔纳米结合底物可以结合和释放DNA/RNA，而不会使其碎裂，从而实现DNA/RNA的完整性(&gt；48 KBP)，与黄金标准的苯酚-氯仿提取相匹配，工艺比珠子和柱子更简单(例如，没有磁铁、高速离心法或试管转移)。此外，纳米结合的结合能力是珠子和柱子的5-30倍。因此，纳米结合实现高DNA完整性的能力与其更高的提取效率以及在一次提取中加载显著更多组织的能力相结合，可以极大地提高分子分析的灵敏度和重复性(即更多质量更高的DNA)。在目标1中，我们将开发一种使用纳米结合底物从FFPE样品中提取高完整性、高产率和无RNA的DNA的方法。在目标2中，我们将开发一种改进的提取方法来从FFPE样本中提取不含DNA的总RNA。在目标3中，我们将通过使用qMSP、qPCR和RT-qPCR进行甲基化、突变和microRNA分析，并与商业柱提取的DNA/RNA进行比较，来验证纳米结合提取的DNA/RNA是否适合于分子图谱。

DESCRIPTION (provided by applicant): Nanobind FFPE DNA/RNA Extraction Despite the growing need for and the demonstrated potential advantages of molecular biomarkers, it has proven difficult to routinely employ them in the diagnosis and management of patients. One reason for this failure has been the logistical challenges of obtaining, rapidly processing, storin, and transporting quick-frozen tissue samples in clinical settings. Standard hospital tissue processing involves fixation in formaldehyde, followed by embedding in paraffin blocks to generate Formalin Fixed Paraffin Embedded (FFPE) tissue samples. If these FFPE samples can be harnessed for routine molecular analysis, the potential for revolutionizing current medical practice exists. FFPE blocks obtained in hospital pathology departments could then be routinely assayed using the newer molecular methods. Moreover, since FFPE samples are usually stored for many years by hospital pathology departments, retrospective molecular evaluations could also be performed, empowering researchers to conduct molecular epidemiologic studies on large cohorts with known clinical outcomes. We aim to develop a new DNA/RNA FFPE extraction method based on a novel and inexpensively fabricated nanomaterial called Nanobind. Nanobind is a thermoplastic substrate containing a hierarchical topography of microscale folds and nanoscale silica flakes. Unlike beads and columns which impart DNA/RNA fragmenting shear forces, the non-porous Nanobind substrate can bind and release DNA/RNA without fragmenting it, achieving DNA/RNA integrity (>48 kbp) that matches gold standard phenol- chloroform extractions with a process that is simpler than beads and columns (e.g. no magnets, high speed centrifugation, or tube transfers). Furthermore, Nanobind has a binding capacity that is 5 - 30 folds greater than beads and columns. Thus, the ability of Nanobind to achieve high DNA integrity combined with its higher extraction efficiency and its ability to load significantly more tissue into a single extraction, could serve to greatly increase molecular assay sensitivity and reproducibility (i.e. more DNA of higher quality). In Aim 1, we will develop a method to extract high integrity, high yield, and RNA-free DNA from FFPE samples using the Nanobind substrate. In Aim 2, we will develop a modified extraction protocol to extract DNA-free, total RNA from FFPE samples. In Aim 3, we will validate the suitability of the Nanobind extracted DNA/RNA for molecular profiling by performing methylation, mutation, and microRNA analysis using qMSP, qPCR, and RT-qPCR and comparing to commercial column extracted DNA/RNA.