The Rockefeller University

Biomarker assay development and quality control assays for studies of exRNA/extracellular nucleic acids.

Purpose

Ribonucleic acid (RNA) is a biomolecule with a variety of function within living cells. It is biochemically very similar to deoxyribonucleic acid (DNA) the biomolecule encoding the genetic information. In contrast to DNA however RNA is less stable and easily degraded by ubiquitiously present enzymes (RNases or more generally nucleases). There is a high nuclease activity in extracellular fluids (biofluids) so that extracellular RNA are commonly viewed as being rapidly degraded and useless for diagnostic applications. But recent research inlcuding in our laboratory showed that at least certain RNA classes are to a certain degree protected from degradation. This is particularly true for miRNAs. We and others have shown that they can be reliably measured in the blood circulation and that a characteristic and robust miRNA signature exists for selected diseases or in pregnancy. However there is very limited data on the biogenesis or the clearance of RNA containing complexes circulating micro RNAs (miRNAs) and almost no data about confounding factors during the sample preparation. Our data on archived clinical samples and publications by other groups indicate that the extracellular RNA content is strongly influenced by blood cell damage variables during samples procurement sample handling as well as by different RNA isolation methods. The use of assays based on distinct technologies and the lack of standardization and precise quantification additionally makes it very hard to compare available results. In this study which follows our pilot study on exRNA isolation for biomarker discovery (IRB KAK-839) we want to identify confounders in sample handling and quantification that can critically influence the circulating RNA profile. In addition we aim to explore techniques for enrichment or concentration of certain RNA classes including but not limited to messenger RNAs (mRNAs) that can be linked to physiological and pathophysiological states. While our focus of this study is on RNA retrieval an characterization a second goal is to direct method development towards optionally extracting extracellular DNA from the same sample after extraction of the RNA an opportinity that discovered unexpectedly when developing our own RNA extraction method. To ensure proper blood collection we will use blood from a cohort of healthy volunteers prepared with different types of anticoagulant and treated with different enzymes and additives after collection. Blood from this cohort will be used for method development in this study and and for quality control purposes in this study and in other studies of extracellular RNA and DNA conducted in this group.