Recent studies indicate that microRNA (miRNA) is contained within exosome. Here 
we sought to optimize the methodologies for the isolation and quantification of 
urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes 
were isolated through ultracentrifugation and characterized by immunoelectron 
microscopy. To determine the RNA was confined inside exosomes, the pellet was 
treated with RNase before RNA isolation. The minimum urine volume, storage 
conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs 
in patients with various kidney diseases was validated with real-time PCR. The 
result shows that miRNAs extracted from the exosomal fraction were resistant to 
RNase digestion and with high quality confirmed by agarose electrophoresis. 16 
ml of urine was sufficient for miRNA isolation by absolute quantification with 
4.15×10(5) copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping 
before stored at -80℃ and was stable in urine when stored at -80°C for 12 
months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The 
detection of miRNA by quantitative PCR showed high reproducibility (>94% for 
intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for 
CKD patients), broad dynamic range (8-log wide) and good linearity for 
quantification (R(2)>0.99). miR-29c and miR-200c showed different expression in 
different types of kidney disease. In summary, the presence of urinary exosomal 
miRNA was confirmed for patients with a diversity of chronic kidney disease. The 
conditions of urine collection, storage and miRNA detection determined in this 
study may be useful for future biomarker discovery efforts.