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Int J Biol Sci 2013; 9(10):1021-1031. doi:10.7150/ijbs.6100 This issue Cite

Research Paper

Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

Lin-Li Lv✉, Yuhan Cao, Dan Liu, Min Xu, Hong Liu, Ri-Ning Tang, Kun-Ling Ma, Bi-Cheng Liu✉

Institute of Nephrology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China.

✉ Corresponding author: Lin-Li Lv, M.D. Ph.D. Email: lvlinliedu.cn. Or Bi-Cheng Liu, M.D. Ph.D. Email: liubc64com. Institute of Nephrology, Zhongda Hospital, School of Medicine, Southeast University. No.87 Ding JiaQiao Road, Nanjing,China 21 More

Citation:
Lv LL, Cao Y, Liu D, Xu M, Liu H, Tang RN, Ma KL, Liu BC. Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery. Int J Biol Sci 2013; 9(10):1021-1031. doi:10.7150/ijbs.6100. https://www.ijbs.com/v09p1021.htm
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Abstract

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. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 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 12months. 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 (R2>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.

Keywords: chronic kidney disease, exosome, miRNA, urine, biomarker.

Citation styles

APA
Lv, L.L., Cao, Y., Liu, D., Xu, M., Liu, H., Tang, R.N., Ma, K.L., Liu, B.C. (2013). Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery. International Journal of Biological Sciences, 9(10), 1021-1031. https://doi.org/10.7150/ijbs.6100.

ACS
Lv, L.L.; Cao, Y.; Liu, D.; Xu, M.; Liu, H.; Tang, R.N.; Ma, K.L.; Liu, B.C. Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery. Int. J. Biol. Sci. 2013, 9 (10), 1021-1031. DOI: 10.7150/ijbs.6100.

NLM
Lv LL, Cao Y, Liu D, Xu M, Liu H, Tang RN, Ma KL, Liu BC. Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery. Int J Biol Sci 2013; 9(10):1021-1031. doi:10.7150/ijbs.6100. https://www.ijbs.com/v09p1021.htm

CSE
Lv LL, Cao Y, Liu D, Xu M, Liu H, Tang RN, Ma KL, Liu BC. 2013. Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery. Int J Biol Sci. 9(10):1021-1031.

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