Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.
Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine.
Diabetes, Cardiovascular and Metabolic Diseases Center, Translational Genomics Research Institute, Phoenix, Arizona 85004, USA.
M Lucrecia
ML
Alvarez
Mahdieh
M
Khosroheidari
Rupesh
R
Kanchi Ravi
Johanna K
JK
DiStefano
D015415:Biomarkers / Q000652:urine; D003928:Diabetic Nephropathies / Q000175:diagnosis / Q000235:genetics / Q000652:urine; D004586:Electrophoresis / Q000379:methods; D063326:Exosome Multienzyme Ribonuclease Complex / Q000235:genetics / Q000302:isolation & purification / Q000652:urine*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000302:isolation & purification / Q000652:urine*; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000652:urine*; D051436:Renal Insufficiency, Chronic / Q000175:diagnosis / Q000235:genetics / Q000652:urine; D014461:Ultracentrifugation / Q000379:methods; D058949:Uromodulin / Q000652:urine
D015415:Biomarkers; D035683:MicroRNAs; D012333:RNA, Messenger; C548612:UMOD protein, human; D058949:Uromodulin; D063326:Exosome Multienzyme Ribonuclease Complex