Nanobind FFPE DNA/RNA Extraction

描述（由申请人提供）：Nanobind FFPE DNA/RNA提取尽管对分子生物标志物的需求不断增长，并且已证明其具有潜在优势，但已证明难以将其常规用于患者的诊断和管理。这种失败的一个原因是在临床环境中获得、快速处理、储存和运输速冻组织样品的后勤挑战。标准的医院组织处理涉及在甲醛中固定，然后包埋在石蜡块中以生成福尔马林固定石蜡包埋（FFPE）组织样本。如果这些FFPE样本可以用于常规分子分析，那么就有可能彻底改变当前的医学实践。在医院病理部门获得的FFPE块可以使用较新的分子方法进行常规分析。此外，由于FFPE样本通常由医院病理部门储存多年，因此也可以进行回顾性分子评价，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，该方法基于一种称为Nanobind的新型廉价制造的纳米材料。Nanobind是一种热塑性基材，含有微米级褶皱和纳米级二氧化硅薄片的分层形貌。与赋予DNA/RNA片段化剪切力的珠和柱不同，无孔Nanobind基底可以结合并释放DNA/RNA而不使其片段化，实现DNA/RNA完整性（>48 kbp），其匹配金标准苯酚-氯仿提取，其过程比珠和柱更简单（例如，没有磁体、高速离心或管转移）。此外，Nanobind的结合能力是珠子和柱子的5 - 30倍。因此，Nanobind实现高DNA完整性的能力，结合其更高的提取效率及其将更多组织加载到单次提取中的能力，可以大大提高分子测定的灵敏度和重现性（即更多的更高质量的DNA）。在目标1中，我们将开发一种方法，使用Nanobind底物从FFPE样品中提取高完整性，高产量和无RNA的DNA。在目标2中，我们将开发一种改进的提取方案，从FFPE样品中提取无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，验证Nanobind提取的DNA/RNA用于分子谱分析的适用性。

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.