Formalin fixed paraffin-embedded (FFPE) tissue archives represent the largest clinically annotated human specimen repository. The estimated millions of available FFPE tissue samples provide vast resource for the discovery of disease epigenetic pathways, biomarkers and drug targets, And yet, these samples are highly underutilized because current methods for extracting molecular information from FFPE samples are slow, labor intensive, insufficiently sensitive to detect gene-bound enzymes (drug targets) and have low throughput to fully exploit their enormous research and clinical potential. There is an unmet need for better tools to take advantage of these archived clinical samples to retrieve chromatin and DNA for the discovery of epigenetic biomarkers for personal medicine. The chromatin immunoprecipitation (ChIP) assay, a widely used approach for identifying genome- associated proteins or DNA modifications, is one of the most powerful tools used in epigenetic studies. To facilitate chromatin analysis, we have developed a high-throughput microplate-based Matrix ChIP-qPCR platform and engineered a device for fast chromatin shearing which uses an array of 96 miniature ultrasound transducers PIXUL (PIXelated ULtrasound). Extraction of chromatin from FFPE samples is a multistep process which includes chromatin shearing. Ultrasound facilitates chromatin extraction from FFPE samples. The goal of this Matchstick Fast Track STTR project (working with UW partner laboratories) is to develop and validate a user-friendly and efficient high throughout PIXUL-based technology, PIXUL-FFPE, to extract and shear chromatin and DNA from small FFPE samples for epigenetic analysis for use in the research market paving the way for use in clinical settings. The following aims are proposed.
In Aim#1 (Phase 1) We will design and validate custom PIXUL 96-well microplate for more efficient extraction of chromatin and DNA from FFPE tissue samples.
In Aim#2 (Phase 2) we will optimize PIXUL-FFPE protocol for fast and high throughput chromatin extraction from small FFPE samples while maximizing sample integrity.
In Aim#3 (Phase 2) we will demonstrate the translational utility of PIXUL-FFPE by comparative ChIP analysis of matched pairs of fresh-frozen and FFPE glioblastoma (GBM) tumor sections.
In Aim#4 (Phase 2) we will modify PIXUL instrument for extraction of chromatin from FFPE samples. Search for epigenetic therapies has slowed down by paucity of available data. Thus, integrating the custom 96-well plates and improved protocol with the modified PIXUL instrument will greatly increase utilization of FFPE specimens in the research market place attracting greater numbers of investigators to explore the vast repositories of archived human tissues and as such advancing precision medicine.
The vast repositories of formalin fixed paraffin-embedded (FFPE) tissue represent enormous resource for discoveries of epigenetic biomarkers, disease pathways and drug targets. And yet, these samples remain largely underutilized because the methods for extraction of chromatin from FFPE for epigenetic analysis are inefficient. We have demonstrated the value of ultrasound for chromatin retrieval from FFPE samples. This proposal addresses the need to develop high throughput and sensitivity technology (ultrasound-based PIXUL-FFPE) for recovery of soluble chromatin from FFPE samples.
