RNA PURIFICATION

1
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), non-coding 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
?
?
Total RNA Yield (ng/ml)
?
Biogenesis of miRNAs (Liu, N et al.,
Developmental Cell 2010)
UF Columns
Exoquick
UF Trizol methods
AIM OF THE STUDY
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.
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.
Bioanalyzer analysis of purified exosomal RNA
MATERIALS METHODS

• EXOSOMES PURIFICATION
• Urinary Exosomes Isolation
• Ultrafiltration, UF (Cheruvanky, A et al., AJRP
  2007)
• Exoquick Reagent (System Biosciences)
• Exosome Size Validation
• Zeta Sizer Nano Range ZS (Malvern)

Urine
UF miRNeasy (87 miRNA)
Exosomes
UF Trizol (80 miRNA)
Flowthrough

• RNA PURIFICATION
• Trizol Method Modifications
• 1 CE IP _at_ RT for 10 min (5T)
• 1 CE IP _at_ -200C for overnight (4T)
• 2 CE IP _at_ -200C for overnight (3T)
• 2 CE IP _at_ -800C for 1hr (2T)
• 1 CE IP _at_ -800C for 1hr (1T)
• CE Chloroform Extraction
• IP Isopropanol Precipitation
• Commercial Columns
• RNeasy kit, Qiagen (1C)
• MiRcury kit, Exiqon (2C)
• RNA mini kit, Ambion (3C)
• miRNeasy kit, Qiagen (4C)
• Seramir kit, SBI (5C)

UF Trizol miRNeasy (93 miRNA)
Exoquick Seramir (67 miRNA)
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
• Urinary exosomes could be efficiently isolated
  by ultrafiltration method as demonstrated by
  light scattering analysis.
• The RNA yield differed substantially between the
  different RNA isolation methods tested.
• Bioanalyzer electropherograms demonstrated the
  presence of miRNAs (ranging in size from 18 to 25
  nt) in the purified exosomal RNA.
• The combination of ultrafiltration for exosomes
  isolation and Trizol-miRNeasy for exosomal RNA
  purification was the most efficient in terms of
  total RNA yield and of miRNA recovery.

• 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