Establishment of Optimal Housekeeping Genes for Urinary Extracellular Vesicle Biomarker Development: A Step Towards Non-Invasive Diagnostics

Background. Urinary extracellular vesicles (UEVs) hold RNAs and can find their application in multiplex biomarker development. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method for gene expression studies. However, there are no reported optimal housekeeping genes available to normalize qPCR data for the UEVs RNA pool.Methods. UEVs were precipitated by Polyethylene glycol (P.E.G. Mn6000) from the urine of 40 human individuals and characterized for their molecular, biophysical, and biochemical purity and integrity. In addition, the expression of five commonly used housekeeping genes B2M, RPL13A, PPIA, HMBS, and GAPDH, were quantified by qPCR in the UEV RNA pool and analyzed through algorithms of NormFinder, geNorm, BestKeeper, and Delta Ct integrated into ReFinder.Results: 12% PEG precipitation yielded round and cup-shaped UEVs. The size and distribution profile of UEVs were around 30 – 100nm through electron microscopy, NTA, and DLS. Acetylcholine esterase and Dipeptidyl peptidase-IV activity were used to arrive at their functional purity. B2M and RPL13A genes were identified as stable genes with a mean stability score of 1.5(geNorm) and below 1 (NormFinder) through ReFinder, which yield comprehensive ranking analysisConclusions: B2M and RPL13A are optimal reference genes and can be used for UEVs based gene expression studies.