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recipient cells, the RNA content of the exosomes may provide million reads apiece.
unique molecular signatures for disease diagnosis and prognosis, Past studies have found strong miRNA representation within
the authors of the new study noted. the exosomal RNA, Wang noted, and the current analysis
An added benefit for those on the hunt for biomarkers is was no exception: some 42 percent of sequence reads in each
the protection that the exosome itself offers against RNA sample and more than three-quarters of those that mapped
degradation, Wang noted. Whereas RNA thats released to the known human sequences came from miRNAs.
directly into the bloodstream or other extracellular sites All told, the team saw 593 different miRNAs (including 185
degrades very quickly, he said, the exosome safeguards RNA new miRNA candidates), with each sample containing be-
over long periods of time, even in harsh conditions. tween 380 and 474 miRNAs. Five miRNAs were particularly
This is a very good criteria for biomarkers, because you common across the samples, making up nearly half of the
dont want to get blood [samples] if RNA is degraded imme- mapped miRNA reads.
diately, Wang said. The exosomal samples also contained a range of other RNA
Such features have contributed to the increasing interest types, from ribosomal RNA and transfer RNA to long, non-
in exosomal RNA biomarkers and in relationships between coding RNA, piwi-interacting RNA, small nuclear RNA, and
extracellular RNA and disease in general (see, for example, small nucleolar RNA.
Gene Silencing News 9/20/2012). Bits and pieces of protein-coding messenger RNAs turned
In addition, several companies have started wading into the up too, though researchers suspect that both the mRNA and
burgeoning exosome field. For instance, System Bioscienc- lncRNA molecules turning up in exosomes are fragments that
es already offers exosomal RNA sequencing services, while may be in the process of degradation particularly since kits
Exosome Diagnostics has been working on potential diagnostic used to produce the sequencing libraries are designed to deal
tests for cancer and other conditions that rely on nucleic acid with small RNAs.
sequences in exosomes found in various biofluids from The use of large-scale RNA sequencing will ensure the
blood to urine (CSN 5/9/2012). discovery and characterization of the whole transcriptome
For the current study, Wang and colleagues set out to better (known and unknown RNAs) of the blood-derived exosomes,
characterize the small RNA content of exosomes in healthy the study authors noted.
blood samples, while at once assessing available methods for A fully characterized transcriptome will help gain a better
preparing the nucleic acids for sequencing. understanding of exosome-mediated molecular mechanisms
To do this, the team first used a reagent called ExoQuick, and will contribute to biomarker discovery, they added. It
from System Biosciences, to isolate the exosomes in blood is expected that the blood-based sequencing assay described
samples from three healthy individuals. here will find clinical applications as a biomarker discovery
The small RNAs in these samples were subsequently pre- tool for disease diagnosis and prognosis.
pared using one of three commercially available kits: the When they compared and contrasted the three small RNA
NEBNext multiplex small RNA library preparation kit; the library preparation methods used in the study, researchers
NEXTflex small RNA sequencing kit from Bioo Scientific; and found that sequence data from samples prepared using each
Illumina TruSeq small RNA sample prep kit. of the kits led to roughly comparable numbers of RNA.
Specifically, researchers used NEB and Bioo Scientific kits But certain RNAs were more or less well represented depending
to prepare two replicate small RNA libraries per person for on the individual tested. There were also subtle differences in the
each of the three individuals tested. The pricier Illumina kit number and nature of exosomal RNAs detected depending on the
was used to prepare two replicate libraries from just one of protocol used.
the participants. In particular, the NEB-prepared samples had the highest pro-
From there, the team sequenced each of the 14 resulting portion of mappable reads overall, on average. These samples
libraries with Illuminas HiSeq 2000, generating nearly 7.3 also had greater representation from new and known miR-
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NAs, the researchers found, particularly those present at in the blood of individuals with cancer, particularly those
relatively low abundance. with later-stage tumors.
NEB had more consistently detectable miRNAs in the Going forward, though, Wang cautioned that those interested
libraries than other kits, Wang said. in using exosomal RNA for diagnostics-related purposes are
Even prior to sequencing, the studys authors noted, quantitative still faced with the challenge of finding the most effective and
PCR analysis of libraries produced using protocols from all three efficient way of isolating the vesicles.
kit developers) indicated that the samples prepared using the NEB For the current analysis, his group relied on chemical isolation
kit had the most robust and reproducible RNA repertoires. using ExoQuick. That approach produces predominantly
At the moment, Wang said he and his colleagues are exosomal samples, he said, though recent analyses suggest
generally using the NEB kits for many of their exosomal that the reagent also precipitates some non-exosomal RNA.
RNA studies, due to its performance in the head-to-head At the moment, the most promising method for obtaining
comparison and its price, which is roughly half that of the pure exosomes appears to be ultra-centrifugation, Wang said.
Illumina TruSeq kit. But because that approach is time-consuming, labor intensive,
Even so, he noted that other kits appear to be beneficial and requires massive amounts of starting material in this
for studies where researchers are interested in exosomal RNA case, blood its generally more suitable for biological studies
expression levels rather than in profiling components of the of exosomes, he said.
exosomal RNA repertoire. Still, in the search for biomarkers, he added that some
The group is already applying exosomal RNA sequencing contamination with non-exosomal RNA should be toler-
to studies of several cancer types. Wang noted that prelim- able, at least in the biomarker discovery phase, provid-
inary, unpublished data suggest that there are detectable ed there are significantly different RNA profiles between
differences in exosomal RNA profiles and/or abundance control and disease samples.
2013 GenomeWeb, LLC. All rights reserved. Unauthorized photocopying or distribution of this copyrighted newsletter is prohibited
by federal law. For permission and information, call 212-651-5632, email reprints@genomeweb.com, or go to www.genomeweb.com.