Abstract

Methods for exRNA Isolation Markedly Impact the Small RNA Expression Profile

Louise Laurent, Associate Professor, University of California-San Diego

The discovery that extracellular RNAs (exRNAs) are present in all tested biofluids has prompted studies into their potential role in cell-cell communication and value as disease-specific diagnostic and prognostic biomarkers. However, such studies require robust and reproducible methods for quantification of exRNAs in biofluids, a task that is made more challenging by the low concentrations of exRNAs in these biological samples. Furthermore, exRNAs are associated with a variety of macromolecular complexes, including extracellular vesicles (EVs), lipoproteins, and ribonucleoproteins, introducing further variability into the isolation of exRNAs. The field has encountered difficulties with validation of findings both within and among different research groups. It is therefore critical to understand the sources of variability that contribute to sub-optimal correlation and lack of reproducibility between different studies. Here, we present results from a study aimed at determining the parameters for various techniques used for isolation of exRNAs from biofluids, and includes comparison of ten exRNA isolation methods used on standardized samples of four biofluids in multiple independent laboratories. The performance of these methods are evaluated by qRT-PCR as well as small RNA sequencing. The results demonstrate that the reproducibility within methods and concordance between methods vary widely, suggesting that the selection of the method used for exRNA isolation is a critical consideration for studies in this field.