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.
Formalin Fixed Paraffin Embedded (FFPE) samples are the standard method for tissue processing in hospital and clinical settings. Developing new techniques for molecular analysis on FFPE samples could revolutionize medical practice and enable powerful retrospective studies on well annotated sample archives. We propose a novel nanomaterial called Nanobind that will extract the highest quality and highest yield DNA/RNA from difficult to process FFPE samples.
|Zhang, Ye; Zhang, Yi; Burke, Jeffrey M et al. (2016) A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction. Adv Mater 28:10630-10636|