1. Volume 86, Issue 2, Pages 433-444 (August 2014)
Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine 
Lesley Cheng, Xin Sun, Benjamin J. Scicluna, Bradley M. Coleman, Andrew F. Hill 
Kidney International 
Volume 86, Issue 2, Pages (August 2014)
DOI: /ki
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2. Figure 1
Work flow representing the isolation of exosomes from urine by differential ultracentrifugation. The steps indicated with black arrows involve isolating exosomes without dithiothreitol (DTT) treatment (-). The work flow indicated by the gray arrows shows exosomes treated with DTT to remove Tamm–Horsfall protein (THP, +).
Kidney International  , DOI: ( /ki )
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3. Figure 2
The characterization of exosomes treated with dithiothreitol (DTT) to deplete Tamm–Horsfall protein (THP). (a) Western immunoblotting of exosomes isolated by ultracentrifugation using Tsg101 and flotillin. Exosomes from SH-SY5Y cells and total cell lysates were loaded as positive controls. (b) Exosomal micro RNA (miRNA) yields quantified by Bioanalyser small RNA assay. The result displayed is the mean value across three independent experiments performed on the same urine sample. Error bars indicate±s.e.m. NS=no significant difference (P=0.18 in a paired two-tail Student’s t-test). (c) Transmission electron microscope (TEM) images of exosome isolations. Arrows indicate the locations of exosomes within the protein network of THP. Bars=200nm, as indicated in the images. Samples were treated with DTT (+) or without DTT (-). (d) Size distribution of exosomes analyzed by the qNano instrument. Exosomes are untreated with DTT.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
Characterization of urinary exosomes by western immunoblotting. (a) Whole urine (WU), cell pellet lysates (Cell), cell-free urine (CF), exosome-depleted supernatant of the 200,000g spin (SN), and exosomes (Exo) (left to right) from the same urine sample were analyzed by immunoblotting with antibodies against exosomal proteins Tsg101, flotillin, and CD63 (under non-reducing conditions), and non-exosomal proteins nucleoporin, GM130, and Bcl2. Exosomes and whole-cell lysates from SH-SY5Y cells were loaded as positive control samples. (b) Bioanalyser chronographs of small RNA profiles obtained from the various urine components as above. (c) Total RNA profile of cell pellets from urine, as determined by an RNA Nano chip.
Kidney International  , DOI: ( /ki )
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5. Figure 4 Study design for comparing different RNA extraction kits.
Kidney International  , DOI: ( /ki )
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6. Figure 5
miRNA yields obtained by different RNA isolation kits. (a) Exosomal miRNA, (b) cell pellet miRNA, (c) cell-free miRNA. Each sample was measured by Bioanalyser small RNA assay in three independent experiments. Percentage of miRNA is obtained by the Bioanalyser, which gates RNA fragments between 10 and 40nt. Two-tailed Student’s t-test was carried out between the two kits. *P=0.001.
Kidney International  , DOI: ( /ki )
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7. Figure 6
A map of chromosomes showing locations of micro RNAs (miRNAs) identified in this study. An example of three highly abundant miRNAs mapped to chromosomes (HG19) based on the coordinates of miRBase version 19. The number of raw reads mapped to the respective miRNA is displayed using Partek Genomic Suites genome browser. NG, Norgen Biotek Urine Exosomal RNA kit; UC, ultracentrifuge.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Mapped known noncoding RNA identified by deep sequencing. (a) Percentage of total reads mapped to noncoding RNA and coding RNA identified by deep sequencing. (b) Venn diagram showing unique and common micro RNAs (miRNAs) in different samples. miRNA with read counts >5 reads per million were shown for comparison.
Kidney International  , DOI: ( /ki )
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9. Figure 8
The presence of the highly abundant micro RNAs (miRNAs) identified across each subject. Heat maps demonstrating the presence or absence of each miRNA identified in cell-free or exosomal preparations across each subject. NG, Norgen Biotek Urine Exosomal RNA kit; UC, ultracentrifuge.
Kidney International  , DOI: ( /ki )
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