Citation:

Chorley, B. Next generation sequencing applications for microRNA biomarker discovery in toxicological studies. SOT, San Antonio, TX, March 11 - 15, 2018.

Impact/Purpose:

This talk is part of a proposed 2018 SOT symposium on the application of next generation sequencing (NGS) to biomarker development for xenobiotic toxicity and exposure. My talk will focus on the current and evolving types of NGS systems and their application to toxicological biomarker discovery, with emphasis on microRNAs and other epigenetic measurements in biofluids. These types of measurements have direct impact on assessing early biological events linked to adverse outcomes (e.g., cancer, liver disease), as well as indicating susceptible subpopulations to environmental exposures - a key element of the recent TSCA reform.

Description:

Next Generation Sequencing (NGS) technology will be reviewed for its base pair resolution, wide dynamic range, and insights into the genome and transcriptome, with special focus upon the biomarker potential of microRNAs (miRNAs). The first part of this presentation reviews commonly used NGS sequencing platforms (Illumina and PacBio) including advantages and disadvantages of each. Attention to sample quality, library preparation, and sequencing depth needed to meet study objectives will be discussed. For RNA sequencing, the transcriptome is generally segregated in two categories: 1) large RNAs like mRNA and long non-coding RNA that are >200 nucleotides in length, and 2) small RNAs of <200 nucleotides, including miRNAs ranging from ~19-25 nucleotides. Special considerations when sequencing differently sized RNAs will be discussed, with emphasis on miRNA discovery. The second part of this presentation will describe the emergence of miRNAs as novel and promising biomarkers. This is due to their cell and tissue specificity that is disrupted during injury and toxicity, and results in the release of these molecules into easily accessible biofluids. miRNAs are stable in these matrices. Further, miRNA sequences and biological functions are largely conserved across species. Taken together, these attributes increase the translational potential of findings generated in experimental systems. Multiple technical obstacles exist such as low levels of detection in biofluids, normalization, assay interference from RNA inhibitors and miRNA contribution from blood cells, and intra- and inter-individual variability. Modified NGS protocols can help overcome some of the technical challenges for application to toxicological assessment and will be discussed utilizing case examples. This abstract does not necessarily reflect EPA policy.

Record Details: