ISOLATION OF URINARY EXOSOMAL miRNAs: COMPARATIVE ANALYSIS OF
DIFFERENT METHODS
Sarath Kiran Channavajjhala1, Marzia Rossato2, Francesca Morandini1, Annalisa Castagna1, Flavia Bazzoni2 and Oliviero Olivieri1
1 University of Verona, Department of Medicine, Unit of Internal Medicine, Verona, Italy
2 University of Verona, Department of Pathology and Diagnostics, Verona, Italy
Quantification of microvescicle size
BACKGROUND
Urinary exosomes are low density and stable membrane vesicles
originating from epithelial cells lining the renal tubules and contain
proteins, lipids, mRNA, miRNA but are devoid of DNA. (Thery, C et al.,
J. Immunol. 2001; Valadi, H et al., Nat. Cell Biol. 2007). miRNAs
comprise a novel class of endogenous, small (20-22 nucleotides), noncoding RNAs that negatively regulate gene expression via degradation or
translational inhibition of their target mRNAs (Mraz, M et al., BBRC 2009
and Weber, J.A et al., Clinical Chemistry 2010).
Urinary microvescicles were isolated by ultrafiltration and analyzed by using
Zeta sizer. The table shows the % of vesicles in the nano size range and the lower
graph represents the size distribution by number of isolated vesicles (n = 4).
Exosomal RNA yield
70

Biogenesis of miRNAs (Liu, N et al., Developmental Cell 2010)
Total RNA Yield (ng/ml)
60
50

40
30

20
10
0
1T
2T
3T
4T
UF + Trizol methods
AIM OF THE STUDY
5T
1C
2C
3C
4C
UF + Columns
5C
Exoquick
To identify and develop a robust and economical method for isolation of
urinary exosomal miRNAs that can be routinely used for the analysis of
miRNAs in different pathological conditions.
Urinary exosomes were isolated using Ultrafiltration (UF) (from 25ml Urine) or
Exoquick Reagent (5 ml urine), total RNA was purified by several methods (refer
to Material and Method section for details) and quantified by using Ribogreen
assay. RNA purification by Trizol (4T), miRNeasy (4C) and Seramir (5C) were
selected for further small RNA analysis.
MATERIALS & METHODS
Bioanalyzer analysis of purified exosomal RNA
EXOSOMES PURIFICATION
Urinary Exosomes Isolation:
• Ultrafiltration, UF (Cheruvanky, A et al., AJRP 2007)
• Exoquick Reagent (System Biosciences)
Urine
Exosomes
UF + Trizol (80% miRNA)
UF + miRNeasy (87% miRNA)
Exoquick + Seramir (67% miRNA)
UF + Trizol+ miRNeasy (93% miRNA)
Exosome Size Validation:
•
Flowthrough
Zeta Sizer Nano Range ZS (Malvern)
RNA PURIFICATION
Trizol Method & Modifications:
• 1 CE + IP @ RT for 10 min
• 1 CE + IP @ -200C for overnight
• 2 CE + IP @ -200C for overnight
• 2 CE + IP @ -800C for 1hr
• 1 CE + IP @ -800C for 1hr
CE – Chloroform Extraction
IP – Isopropanol Precipitation
Commercial Columns :
• RNeasy kit, Qiagen
• MiRcury kit, Exiqon
• RNA mini kit, Ambion
• miRNeasy kit, Qiagen
• Seramir kit, SBI
(5T)
(4T)
(3T)
(2T)
(1T)
Exosomal RNA isolated with the indicated methods was analyzed using RNA Nano
6000 Kit in an Agilent 2100 Bioanalyzer. The electropherograms show the size
distribution in nucleotides (nt) and fluorescence intensity (FU) of total RNA
purified from exosomes. The peak at 4 nt is an internal standard.
CONCLUSIONS
(1C)
(2C)
(3C)
(4C)
(5C)
RNA QUANTIFICATION
• Ribogreen for the quantification of single stranded RNA (total RNA yield)
• Agilent Bioanalyzer for the evaluation of RNA integrity and the presence of small
RNA fraction
1. Urinary exosomes could be efficiently isolated by ultrafiltration method
as demonstrated by light scattering analysis.
2. The RNA yield differed substantially between the different RNA
isolation methods tested.
3. Bioanalyzer electropherograms demonstrated the presence of miRNAs
(ranging in size from 18 to 25 nt) in the purified exosomal RNA.
4. The combination of ultrafiltration for exosomes isolation and TrizolmiRNeasy for exosomal RNA purification was the most efficient in terms
of total RNA yield and % of miRNA recovery.