For what factors should we normalize urinary extracellular mRNA biomarkers?

Abstract

mRNA is a critical biomolecule involved in the manifestation of the genetic code into functional protein molecules. Its critical role in the central dogma has made it a key target in many studies to determine biomarkers and drug targets for numerous diseases. Currently, there is a growing body of evidence to suggest that RNA molecules around the size of full-length mRNA transcripts can be assayed in the supernatant of human urine and urinary extracellular mRNA could provide information about transcription in cells of urogenital tissues. However, the optimal means of normalizing these signals is unclear. In this paper, we describe relevant first principles as well as research findings from our lab and other labs toward normalization of urinary extracellular mRNA.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

“…Moreover, prolonged collection of uEVs may accelerate their degradation (Oosthuyzen et al., 2013), although this remains under debate (Mitchell et al., 2009). The measurement of an absolute excretion rate of a urine biomarker using a timed collection can be approximated in a spot urine measurement by a ratio to urinary creatinine (Gunasekaran et al., 2019), which has been shown to be highly effective for both intra‐individual comparison (96% of uEV variation explained by creatinine concentration) and inter‐individual comparison (47–82%) (Blijdorp et al, 2021). Creatinine is a waste product of muscle catabolism.…”

“…Use of uEVs from existing biobanks also represents a clinical challenge because the standardization necessary for many assays may be insufficient or different compared with what is needed for uEV assays. An additional challenge in the field relates to normalizing biomarker signals (Gunasekaran et al., 2019) because urine is one of the most dynamic biofluids. In order to move the field of uEV research forward, uEV reference standards are needed for many experimental purposes, including single EV analysis, for example, for flow cytometry and particle analysers for assessment of size and concentration or normalization to excretion rate and uEV processing‐related variation.…”

CD44v8-10 mRNA contained in serum exosomes as a diagnostic marker for docetaxel resistance in prostate cancer patients

“…cDNA was synthesized in a total reaction volume of 20 μL using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). After determination of RNA concentration, 300 ng of cellular total RNA was used in the reaction, whereas the whole amount of exosomal total RNA was subjected to cDNA synthesis in accordance with previously published methods [18,21,22]. Digital PCR (dPCR) was performed using 1.5 μL of the cDNA sample according to the manufacturer's instruction (Thermo Fisher Scientific).…”

A Method to Analyze Urinary Extracellular Vesicles

“…95 The normalization of the input to the volume of sample input is also recommended. 96 Moreover, the evaluation of this approach relies on the probe or primers used which are limited. Microarray genomic analysis is another technology that offers a way to assess thousands of transcripts in UEVs using a gene expression array.…”