Normalizing Urinary Extracellular Messenger RNA Biomarkers: Theoretical Considerations and a Review of Experimental Findings

Abstract
Messenger RNA (mRNA) has been extensively annotated, and its crucial role in the central dogma has made it a key target in many studies of biomarkers and drug targets. Extracellular vesicles shuttle mRNA, among other molecular cargo. mRNA in urinary extracellular vesicles has potential as a biomarker, particularly in diseases affecting cells of the urothelial tract. There is evidence that this mRNA could provide information about transcription in cells of urogenital tissues. However, the optimal means of normalizing these signals is unclear. In the more common cell lysate context, gene expression can be normalized to robustly expressed genes that have similar expression between cells. However, in the context of extracellular mRNA shuttled by extracellular vesicles, normalization strategies remain undefined. This talk will describe relevant first principles as well as research findings from our lab and other labs toward normalization of urine extracellular mRNA. The talk will focus on at least two hypothetical sources of confounding that might be important denominators when normalizing assays of urinary extracellular mRNA transcripts. The first is changes in the composition of the biofluid matrix broadly affecting analytes’ concentrations (i.e., intra-individual changes or inter-individual differences in urine composition due to having recently consumed water). The second is broad changes or differences in the expression of genes without direct relevance to the specific biology of interest. The extent to which each of these factors must be taken into account during normalization will depend on the magnitude of target gene’s expression signal in the disease relative to the magnitude of the noise created by these potentially confounding factors. The rationale for the use of urinary creatinine as the traditional reference molecule for many urinary assays will be discussed, as will advantages and disadvantages of urinary creatinine for normalization of urinary extracellular mRNA. In addition, the possibility of using reference genes to normalize assays of urinary extracellular mRNA will be discussed. Prior experiments bearing on the feasibility of these approaches will be reviewed. The talk will also discuss other potential strategies for normalization, such as normalizing to the concentration of extracellular vesicles. Finally, the talk will attempt to synthesize these ideas and the findings from the literature into suggestions for those developing urinary extracellular mRNA assays.