title: Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles.
abstract: Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases.
authors.affiliation: Hitachi Chemical Research Center, Inc., Irvine, California, United States of America.
authors.forename: Taku
authors.initials: T
authors.lastname: Murakami
authors.affiliation: Hitachi Chemical Research Center, Inc., Irvine, California, United States of America; Genomics High-Throughput Facility, University of California Irvine, Irvine, California, United States of America.
authors.forename: Melanie
authors.initials: M
authors.lastname: Oakes
authors.affiliation: Hitachi Chemical Research Center, Inc., Irvine, California, United States of America.
authors.forename: Mieko
authors.initials: M
authors.lastname: Ogura
authors.affiliation: Hitachi Chemical Research Center, Inc., Irvine, California, United States of America.
authors.forename: Vivian
authors.initials: V
authors.lastname: Tovar
authors.affiliation: Hitachi Chemical Research Center, Inc., Irvine, California, United States of America.
authors.forename: Cindy
authors.initials: C
authors.lastname: Yamamoto
authors.affiliation: Hitachi Chemical Research Center, Inc., Irvine, California, United States of America; NanoSomiX, Inc., Irvine, California, United States of America.
authors.forename: Masato
authors.initials: M
authors.lastname: Mitsuhashi
mesh_terms: D001681:Biological Assay / Q000379:methods*; D055354:Exosomes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007678:Kidney Glomerulus / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D009928:Organ Specificity; D012333:RNA, Messenger / Q000235:genetics / Q000652:urine; D012015:Reference Standards; D012492:Salts / Q000737:chemistry; D014461:Ultracentrifugation
chemical_list: D012333:RNA, Messenger; D012492:Salts