Novel Methods for Obtaining Molecular Information from Archived Tissue Samples (R43/R44)

With the advent of numerous next-generation sequencing technologies and other enhanced "omics" approaches, there are significant opportunities for measuring transcriptional, epigenetic, proteomic, or metabolomic alterations in frozen or formalin-fixed, paraffin-embedded (FFPE) archived tissue samples collected and stored from clinical, environmental health, and toxicology studies. There exist numerous archives containing tissues and biological samples obtained from both animal toxicology and human clinical studies. In some cases, there is extensive pathology information on these samples, and to a more limited extent, exposure and lifestyle data or phenotype information. However, because of technical limitations, measuring molecular changes that correspond to pathology or other phenotypic endpoints has been a significant challenge.

To maximize the use of archived samples from human clinical studies and animal toxicology studies, improvements are needed in methods to extract protein, DNA, RNA, and metabolites from both FFPE tissues and frozen biological samples. Also needed is the development of new methods and reagents to maintain high-quality DNA, RNA, protein, and small molecules during collection and storage of biological samples. In some cases, blood or other biological samples need to be preserved during the 24-48 hour transport from the field prior to refrigeration or freezing.  Therefore, this FOA supports the development of methods and tools that enable next-generation sequencing and other "omics" analyses of biological fluids or frozen or FFPE tissues.

Research topics include:

• Novel methods and reagents for extraction/enrichment of methylated DNA or post-translational modifications of histones from FFPE tissues.
• Development of a DNA adductome panel (DNA adducts).  The panel should assess covalent binding of endogenous or exogenous chemicals to DNA that reflects exposure to DNA-damaging agents. Adducts may encompass direct binding of chemicals or exogenous agents to DNA or adducts from metabolic processes resulting from response to exposure to environmental toxicants.
• Improved methods for micro RNA (miRNA) extraction and enrichment from preserved tissues and biofluids for genome-wide miRNA analysis and development of distinguishing markers for intracellular versus extracellular miRNA from preserved tissues and biofluids.
• New purification/separation methods without differential centrifugation to isolate exosomes from blood, culture media or other biofluids, focusing on exosomes containing miRNA, mRNA, DNA or protein suitable for molecular analysis that reflect exposures to environmental toxicants.
• Agents to maintain integrity of extracellular and intracellular metabolites and other small molecules for metabolite profiling studies.
• Development of cost-effective alternative reagents for tissue fixation (i.e., to replace formalin), processing, and high-temperature paraffin embedding for preservation of nucleic acids and proteins while maintaining optimal tissue morphology for histopathology evaluation.
• Development of methods for preservation of blood or other biological samples for 24-48 hour transport from the field prior to refrigeration or freezing and demonstration of use of preserved samples versus frozen samples in molecular analysis.  
• Devising and testing methods to standardize metrics that assess the quality and quantity of nucleic acids, proteins or small molecules (metabolites) in archived FFPE tissues stored for variable periods of time (months to years).

For methods developed for FFPE samples, the use of fresh or frozen tissues as a basis of comparison for omics analysis is encouraged. 

See Section VIII. Other Information for award authorities and regulations.