the complete exosome workflow solution: from isolation to … · 2016. 5. 14. · 10 30 50 70 90...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 253957 15 pageshttpdxdoiorg1011552013253957

Research ArticleThe Complete Exosome Workflow Solution From Isolation toCharacterization of RNA Cargo

Jeoffrey Schageman Emily Zeringer Mu Li Tim Barta Kristi Lea Jian GuSusan Magdaleno Robert Setterquist and Alexander V Vlassov

Life Technologies Austin TX 78744 USA

Correspondence should be addressed to Alexander V Vlassov sashavlassovlifetechcom

Received 15 April 2013 Revised 26 July 2013 Accepted 26 July 2013

Academic Editor Chung-Liang Chien

Copyright copy 2013 Jeoffrey Schageman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Exosomes are small (30ndash150 nm) vesicles containing unique RNA and protein cargo secreted by all cell types in culture They arealso found in abundance in body fluids including blood saliva and urine At themoment themechanismof exosome formation themakeup of the cargo biological pathways and resulting functions are incompletely understood One of their most intriguing rolesis intercellular communicationmdashexosomes function as the messengers delivering various effector or signaling macromoleculesbetween specific cells There is an exponentially growing need to dissect structure and the function of exosomes and utilize themfor development of minimally invasive diagnostics and therapeutics Critical to further our understanding of exosomes is thedevelopment of reagents tools and protocols for their isolation characterization and analysis of their RNA and protein contentsHere we describe a complete exosome workflow solution starting from fast and efficient extraction of exosomes from cell culturemedia and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generationsequencing techniques Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived fromHeLacell culture media and human serum using Ion Torrent PGM as a sequencing platform

1 Introduction

Exosomes are a type of vesicle 30ndash150 nm in size that havereceived increased attention over the past few years [1ndash7]Exosomes are secreted by all cell types in culture and alsofound naturally in blood urine cerebrospinal fluid breastmilk saliva ascitic fluid and amniotic fluid in very highnumbers [7 8] Depending on the cell of origin manydifferent functions have been attributed to exosomes such asmorphogen transporters in the creation of polarity duringdevelopment and differentiation [4] role in programmedcell death angiogenesis inflammation coagulation [9] andmigration of Dictyostelium cells by the secretion of chemoat-tractant signals [10] Valadi et al [11] demonstrated thatMC9and HMC-1 mast cells secrete exosomes that contain mRNAfrom approximately 1300 genes and small RNAs including121 unique microRNAs The transfer of exosomes to a donorcell showed that at least some mRNAs were full length asthey were translated in the recipient cell Glioblastoma cells

also secrete exosomes and microvesicles containing mRNAmiRNA and angiogenic proteins [12] When taken up byhost human brain microvascular endothelial cells mRNAmolecules were translated and tubule formation by the targetendothelial cells was stimulated The spread of oncogenesby exosomes and microvesicles secreted by tumor cells hasbeen reported [13] Exosomes also play a crucial role indisseminating pathogens such as prions and viruses from onecell to another [14ndash17] Interest towards exosomes from theirfunction in the body to more practical applications such asthe use in diagnostics (based on analysis of their RNA andprotein content) and therapeutics development has grownexponentially in the last five years [18ndash20]

Despite a rapidly growing number of research studiesthere is still rather limited and superficial information avail-able regarding RNA content of exosomes [21] Several reportshave been published to date utilizing sequencing technolo-gies to characterize the RNA content of exosomes derivedfrom THP1 and HUVEC cells [22] DC cells [23] hES-MSC

2 BioMed Research International

cells [24] placenta [25] and breast milk [26] Except forNolte-rsquot Hoen et al [23] who characterized awide populationof small RNA residing within the vesicles all studies solelyfocus on analysis of the miRNA content Significantly lessinformation is available regarding other RNA classes andother types of body fluids such as blood Moreover certainreports contain a number of contradictions Several papersfor example indicate that the RNA ldquocargordquo of exosomes issubstantially different from the parental cell content [12 2728] This runs counter to several other authors working withcancer cells who have noted that themiRNA content for theiroriginating cancer cells is similar to that found in circulatingexosomes opening up the possibility that exosomes could beused as unique diagnostic markers [29 30]The complicatingfactor between these studies is a lack of standardized tech-niques protocols and workflows for isolation of exosomesand downstream analysis of their constituents Currently themost popular approach for isolation of exosomes is basedon ultracentrifugation [31] which allows the researcher toobtain highly pure exosomes however it is a very lengthydifficult and unreliable process To address the urgent needto have a simple solution for isolation of exosomes severalreagents were developed by biotechnology companies in thelast two years System Biosciences released a proprietaryreagent named ExoQuick that can be added to serumconditioned cell media or urine and is claimed to precipitatethe exosomes [32] HansaBioMed is offering an array ofproducts called ExoTest kitsmdashfeaturing anti-CD63 -CD81or -CD9 antibodies immobilized on 96-well platesmdashforexosome capturing and characterization [33] Bioo Scientificlaunched the ExoMir kit that essentially removes all cellsplatelets and cellular debris on one microfilter and capturesall vesicles bigger than 30 nm on a second microfilter usingpositive pressure to drive the fluid [34] One should keep inmind that depending on the isolation procedure exosomepreparations are contaminated to a various extent with othermicrovesicles or RNA-protein complexes [35] dramaticallyaffecting the outcome of downstream analysis

Here we describe a complete exosome workflow solutionstarting from fast and efficient extraction of exosomes fromcell culture media and serum to robust isolation of RNA andcharacterization of exosomal RNA content using qRT-PCRand next-generation sequencing techniques Effectivenessof this workflow is demonstrated by analysis of the RNAcontent of exosomes derived from HeLa cells and bloodserum using Ion Torrent PGM as a sequencing platformThis straightforward standardized workflow can be utilizedfor analysis of the exosomal RNA cargo shedding somelight on the mechanisms of formation preferential loadingand functions of these fascinating vesicles In a similarfashion disease-specific RNA signatures residing withinthe exosomes can be uncovered and potentially used fordevelopment of minimally invasive or noninvasive diag-nostics providing the possibility of someday being able toanalyze RNA ldquocontentrdquo of organs and tissues without biopsy

2 Materials and Methods21 Materials Total exosome isolation (from serum) reagent(Invitrogen) Total exosome isolation (from cell culturemedia) reagent (Invitrogen) Total exosome RNA and proteinisolation kit (Invitrogen) blood serum from two donorscell culture media from HeLa cells 10x PBS nuclease-freewater (Ambion) 100 ethanol nonoptical adhesive cov-ers (Applied Biosystems) optical adhesive covers (AppliedBiosystems) 384-well PCR standard plates (Applied Biosys-tems) 96-well PCR standard plates (Applied Biosystems)universal PCR master mix II (Applied Biosystems) humanTaqMan miRNA assays Veriti 96-well thermocyclers (Ap-plied Biosystems) 7900HT Instrument SW v23 TaqManmicroRNA reverse transcription kit (Applied Biosystems)1000 reactions and Ion Total RNA-Seq kit v2 (Life Technolo-gies) were utilized

22 Extraction of Exosomes from Serum and Cell Media UsingTotal Exosome Isolation Reagents

221 Cell Culture Media Fresh cell media was harvestedfrom HeLa cells grown in T175 flasks Initially the cells weregrown in media containing 10 FBS (to sim90 cell density)then washed twice with PBS and grown for the remaining12 h in 10 exosome-depleted FBS The cell media sampleswere then centrifuged at 2000 g for 30min to remove celldebris The supernatant containing the cell-free cell mediawas transferred to a fresh container and held on ice until useNext each sample was combined with 12 volume of Totalexosome isolation (from cell media) reagent and mixed wellby vortexing or pipetting up and down until a homogenoussolution was formed Typical cell media volume utilizedwas 1mL however the range of 100 120583Lndash50mL was useddepending on the downstream applicationThe samples wereincubated at 4∘C overnight and then centrifuged at 4∘C at10000 g for 1 hThe supernatant was aspirated and discardedand the exosome pellet was resuspended in PBS buffer andthen stored at 4∘C short term (1ndash7 days) or minus20∘C long term

222 Human Blood Serum Frozen serum samples werethawed in a water bath at room temperature until sampleswere completely liquid and then centrifuged at 2000 gfor 30min to remove any cellular debris The supernatantcontaining the cell-free serum was transferred to a freshcontainer and briefly held on ice until use Next each serumsample was combined with 15th volume of Total exosomeisolation (from serum) reagent and then mixed well byvortexing or pipetting up and down until a homogenous solu-tion was formed Typical serum volume utilized was 100 120583Lhowever the range of 50 120583Lndash5mL was used depending onthe downstream application The samples were incubated at4∘C for 30min and then centrifuged at room temperatureat 10000 g for 10min The supernatant was aspirated anddiscarded and the exosome pellet was resuspended in PBSbuffer and then stored at 4∘C short term (1ndash7 days) or minus20∘Cfor long term

BioMed Research International 3

23 Sizing and Quantification of Exosomes with NanosightLM10 Instrument Exosomes purified from cell media andblood serumwere diluted with PBS buffer (10ndash5000x in orderto have the nanovesicle concentration in the working rangefor the Nanosight LM10 2 times 108ndash8 times 108) and then quantifiedand sized using the Nanosight LM10 instrument (NanosightUK) following the manufacturerrsquos protocol The LM10 usesa laser light source to illuminate nanoscale particles (10ndash1000 nm) which are seen as individual pointscatters movingunder Brownian motion The paths of the point scattersor particles are calculated over time to determine theirvelocity which can be used to calculate their size independentof density The image analysis NTA software compiles thisinformation and allows the user to automatically track thesize distribution and number of the nanoparticles

24 Western Blot Analysis Exosome samples isolated fromcell media or blood serum (typically equivalent of 50120583L cellmedia and 5 120583L serum)weremixedwith 2x nonreducingTris-glycine SDS sample buffer (Novex) for CD63 and 2X reduc-ing buffer for CD9 then heated at 75∘C for 5min and loadedonto a 15mm times 15 well 4ndash20 Tris-Glycine gel (Novex)Benchmark prestained protein ladder (Invitrogen) was addedto one well as a control to monitor the molecular weight ofthe protein samples The gel was run under denaturing con-ditions at 150V for 15 h and then transferred to a membraneusing the iBlot instrument (Life Technologies) After transferthe membranes were processed on the BenchPro 4100 (LifeTechnologies) with CD63 or CD9 antibody (Abcam) diluted100 120583g into 20mL The WesternBreeze Chemiluminescencekit was utilized on the next step membranes were exposed toX-ray film for 1ndash10min and the film was analyzed

25 RNA Recovery Using the Total Exosome RNA and ProteinIsolation Kit The Total exosome RNA and protein isolationkit (Invitrogen) was utilized for recovery of RNA from theexosome samples obtained with the reagent and ultracen-trifugation protocol and parental samples for each sampletype HeLa cell pellets (1 times 106 cells) and cell-free serum200120583L of each sample (brought up to volumewith PBS if nec-essary) was combined with 205120583L of 2x denaturing solutionvortexed to lyse and then incubated on ice for 5min Afterincubation 410120583L of acid-phenol chloroform was added tothe mixture and vortexed for 30ndash60 sec to mix Samples werethen centrifuged for 5min at 10000 g at room temperature toseparate the mixture into aqueous and organic phases Oncecentrifugation was complete the aqueous (upper) phase wascarefully removed without disturbing the lower phase or theinterphase and transferred to a fresh tube

125 volume of 100 EtOH was added to the aqueousphase for each sample and then vortexed to mix 700 120583L ofvolume was placed onto spin column in a collection tube andthen spun at 10000 g for 15 sec to move the sample throughthe filter cartridge Samples were then washed once with700120583L wash solution 1 and 2x with 500120583L wash solution23 (centrifuged at 10000 g for 15 sec for each wash) Afterwashing filter was dried by spinning for an additional 1minat 10000 g The filter cartridge was transferred into a fresh

collection tube and 50120583L of preheated (95∘C) nuclease-freewater was applied to the center of the filter Samples werecentrifuged for 30 sec at 10000 g to recover the RNA andthen a second 50120583L volume of preheated (95∘C)nuclease-freewaterwas applied to the center of the filter and centrifuged for30 sec at 10000 g After the second spin the eluate containingthe RNA was collected and stored at minus20∘C For cell pelletRNA a DNase treatment was performed using the DNase-free Kit (Ambion) to remove any contaminatingDNADNasetreatment was not performed on exosome samples as theyhad a much smaller input After treatment each sample wasdiluted to 2 ng120583L and 1 120583L analyzed on the Agilent 2100Bioanalyzer using the Agilent RNA 6000 Pico Kit (SeriesII) to determine the mass of RNA going into downstreamanalysis

26 Reverse Transcription and Quantitative Real-Time PCR(qRT-PCR) Analysis of the RNA Sequences Isolated fromthe Exosomes Reverse Transcription (RT) Master Mix wasprepared for each sample using the TaqMan MicroRNAReverse Transcription Kit reagents and protocol (AppliedBiosystems) with gene specific RT primers for five miRNAtargets (miR16 miR24 miR26a miR451 and let7e) Ten 120583Lof the RT master mix was added to corresponding wells ina 96-well plate and 5120583L of each sample was added to themaster mix Plates were covered with adhesive (nonoptical)cover and spun down to remove air bubbles and then placedinto a 9700 thermocycler and incubated as follows 4∘C for5min 16∘C for 30min 42∘C for 30min and 85∘C for 5minReactions were kept at 4∘C until use

qPCR master mixes were prepared for each of fivemicroRNAs by combining 5 120583L of AB Universal PCR MasterMix II 25 120583L of nuclease-free water and 05 120583L of the 20xTaqMan assay After mixing 8 120583L of each master mix wasplaced into wells in a 384-well plate (enough for triplicatereactions for each isolation replicate) Two 120583L of each RTreaction was added in triplicate to the master mix of eachtarget and the plates were sealed with an optical adhesivecover Plates were spun down to remove air bubbles andthen placed into a 7900HT instrument and run using thefollowing thermocycler protocol 95∘C for 10min + (95∘C for15 s 60∘C for 60 s) for 40 cycles Once the run was completeautomatic Ct analysis was performed with SDS v23 softwareand average and standard deviations were calculated for eachset of isolations and qPCR reactions for each target

27 Preparation of the Small RNA Libraries and SequencingExosomal RNA Small RNA libraries were prepared usingthe Ion Total RNA-Seq Kit v2 (Life Technologies) protocoland materials However a number of modifications wereintroduced into the RNA-Seq protocol in order to accommo-date the specific nature of the exosome samples (1) relativelylow amount of RNA and (2) majority of the RNA cargobeing lt200 nt in size For library construction the RNAsample was dried down to 3 120583L and then combined with thehybridization reagents and incubated at 65∘C for 10min and16∘C for 5min Ligation reagents were then added and thesamples were incubated at 16∘C for 16 h (overnight) After


ligation reverse transcription was performed RTmaster mixwas added to the samples tubes were incubated at 70∘C for10min samples were snap-cooled on ice the RT enzymewas added and the samples were incubated at 42∘C for30min cDNA from the RT reaction was purified using thekitrsquos clean-up module containing MagMAX Beads (5 120583L perwell of a 96 well plate) and eluted in 12 120583L of nuclease-free water Six 120583L of the purified cDNA was combined withPCR primers and Platinum PCR SuperMix High Fidelityreactionmixwas then placed in a thermocycler and amplifiedusing the following protocol 94∘C for 2min (94∘C for 30 s50∘C for 30 s and 68∘C for 30 s) 2 cycles (94∘C for 30 s62∘C for 30 s and 68∘C for 30 s) 16 cycles 68∘C for 5minOnce protocol was complete reactions were stored on iceuntil purification The amplified DNA (final library) foreach sample was purified using the kitrsquos clean-up modulecontaining MagMAX Beads (5120583L per well of a 96-well plate)and eluted in 10120583L of nuclease-free water Final libraries werestored on ice for the short term and at minus20∘C for long termTo assess the yield and size distribution 1 120583L of the librarywas run on an Agilent DNA High Sensitivity chip (Agilent)The molar concentration of the library was determined withthe Agilent 2100 Bioanalyzer Instrument Expert softwareand used to dilute libraries to correct concentration forsequencing Sequencing was performed for each sample onthe Ion Torrent PGM instrument using 318 chips (11000000wells per chip) and the protocol listed in the Total exosomeRNA and protein isolation kit (Invitrogen) with 160 flows (40cycles)

28 Sequence Data Acquisition and Preprocessing Uponcompletion of each PGM sequencing run the TorrentSuite software (httpioncommunitylifetechnologiescomdocsDOCS-7189) performed base calling from raw signalsReads from polyclonal beads and low quality reads werefiltered out and 31015840 adapter sequences were trimmed beforethe sequence was output into a FASTQ formatted filefor analysis (httpenwikipediaorgwikiFASTQ format)Contained within this file are the sequences (reads)which correspond to a single bead location on theIon 318 chip and per base PHRED scaled [36] (httpenwikipediaorgwikiPhred quality score httpioncom-munitylifetechnologiescomdocsDOC-2306) quality val-ues

With the data in the FASTQ format it was entered intothe exosome-seq mapping pipeline for final analysis (seeFigure 4) While the Torrent Suite trims 31015840 adapter and filterslow quality reads quite effectively some residual suboptimalsequences occasionally remain For this reason the first stepin the pipeline includes 31015840 quality and adapter trimming usingthe FASTX-Toolkit (httphannonlabcshledufastx toolkit)and Cutadapt [37] programs respectivel Specifically theFASTX-Toolkit subprogram fastq quality trimmer scans eachread and its respective quality values from the 51015840 to the 31015840end of the reads and trims bases below a PHRED qualityvalue of 17 If the read length falls below 17 bases in lengthit is removed from further processing This is to reducethe number of ambiguously mapped reads which become

more frequent as reads get shorter The remaining reads arescanned for P1 adapter sequences at the 31015840 end using cutadaptAgain if the read length falls below 17 bases after adaptertrimming it is removed from analysis

These preprocessing steps are critical since low qualitysequence and 31015840 adapter sequences can be a source ofmisalignment (noise) or preclude the read from mapping atall which causes problems when trying to map reads to thelocations in the genome from which they are transcribed

29 Data Analysis Quality Control and Assessment Afterpreprocessing each read of the FASTQ file was examinedbased on global quality metrics using fastqc httpwwwbioinformaticsbbsrcacukprojectsfastqc The result-ing graphs and statistics were based on quality valuesnucleotide composition sequence length and most highlyrepresented sequences (k-mers) for each libraryThis analysisprovided a high-level evaluation of sequence quality andpotential biases which could be present in a given RNA-Seqsample If irregularities are observed such as quality valuedistributions shifted lower this may be an indication that aproblem has occurred upstream from the analysis pipeline

210 Data Analysis Iterative Mapping As mentioned ear-lier the goal of the sequencing and analysis is to deter-mine measurable global expression profiles using multi-ple reference datasets The alignment steps in this work-flow utilize the SHRiMP2 [38] aligner to map the FASTQsequences to miRBase precursors The SHRiMP2 alignerwas chosen as it has been tested and optimized specifi-cally for mapping miRNA sequences using special param-eters provided by the authors and can be implementedto align longer sequences with high sensitivity The align-ment algorithm used attempts to optimally align each readsequence to a reference containing miRNA hairpins (miR-Base build 180) tRNA and ribosomal RNAs and thenreport each alignment in a sequence alignmentmap (SAM)(httpsamtoolssourceforgenetSAM1pdf) formatted fileThis file format is the current standard alignment format andnumerous downstream analysis tools have been written touse it for easy parsing and counting of reads to individualtranscripts

Previously published reports [11 39] have shown thatmRNAs are also found in isolated exosome fractions andmaybe effectively quantitated through sequencing To examinemRNA levels in exosomes compared to parental cell samplesa secondary mapping step was completed using unmappedreads from the previous miRNA mapping step as input foralignment toRefSeq (build Jan 2012) transcripts themajorityof which are protein coding mRNAs

Finally in order to comprehensively account for addi-tional known RNA species which could be present in exo-some fractions the reads that did not map in the RefSeqstep were taken andmapped to the noncoding RNA databaseNoncode (v20) [40]This step allowed for the quantitation ofpiRNA scaRNA and snoRNAs not previously annotated byRefSeq


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Figure 1 Both ultracentrifugation protocol and Total exosome isolation reagent enable recovery of very clean population of exosomes fromcell media and serum ((a) and (b)) Analysis of exosomes recovered fromHeLa cell media using the Total exosome isolation reagent (from cellculture media) and ultracentrifugation protocol by Nanosight LM10 instrument ((c) and (d)) Analysis of exosomes recovered from serumusing the Total exosome isolation reagent (from serum) and ultracentrifugation protocol by Nanosight LM10 instrument

To determine read counts matching each reference RNAat each step a simple Perl scriptwaswritten to scan each of thethree SAM files and tally mapped read counts per transcriptand then report them in tabular format Once complete thesecount tables were used to compare enrichment of particulartranscript across sample types

3 Results and Discussion

31 Isolation and Initial Characterization of Exosomes fromCell Media and Serum The original approach for isolation ofexosomes still widely used is based on ultracentrifugationin combination with sucrose density gradients or sucrosecushions to float the relatively low-density exosomes awayfrom other vesicles and particles [31] These protocols rangein time from 8 to 30 h and require an ultracentrifuge andextensive training to ensure successful isolation of exosomesIn addition only six samples can be processed at a time andthe sample volume needed is quite large (typically 10ndash30mL)As for a way to enable simple and fast exosome isolationwe developed two Total exosome isolation reagents Thesereagents allow straightforward and reliable recovery of fully

intact exosomes from cell culture media and blood serumsamples in a wide volume range and are suitable for highthroughput applications By tying up water molecules thereagents force less-soluble components such as nanovesiclesout of solution To isolate exosomes the reagent is addedto a biological sample and the mixture is incubated at 4∘Cfollowed by precipitation through the standard centrifugationat 10000 g The pellet containing the exosomes is thenresuspended in PBS or similar buffer and the exosomes areready for the end-point analysis or biological studies on theirpathways and functions

We isolated exosomes from HeLa cell culture media(chosen as a model system as its use is ubiquitous in thelaboratory and since a general understanding of this systemhad been achieved) and blood serum samples (derivedfrom healthy human donors) using Total exosome isolationreagents as well as the ultracentrifugation procedure [31]for comparison Sizing and quantification of exosomes wereperformed with the NanoSight LM10 instrument and resultsare shown in Figures 1(a) and 1(b) (cell media) and Figures1(c) and 1(d) (serum) All nanoparticles recovered with bothprotocols were smaller than 300 nm most of them being inthe typical exosome size range of 30ndash150 nm


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(d)

Figure 2 Western blot analysis for the presence of exosomal marker proteins CD63 and CD9 in HeLa cell culture media ((a) (b)) and serum((c) (d)) derived samples Exosomes isolated with either the Total exosome isolation reagents or ultracentrifugation protocols were separatedon 4ndash20Tris-Glycine gels StandardWestern blot procedures with anti-CD63 and anti-CD9 antibodies were used to detect exosomal proteinmarkers

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Figure 3 Analysis of the exosomal miRNA levels in HeLa cell culture media (a) and serum preparations (b) by quantitative RT-PCR RNAwas isolated using the Total exosome RNA and protein isolation kit from exosomes extracted using the Total exosome isolation reagents andthe ultracentrifugation protocols Levels of five microRNAs (miR16 miR24 miR26a miR451 and let7e) were quantified by qRT-PCR usingTaqMan assays and reagents

Both the reagent and ultracentrifugation proceduresallowed recovery of a significant number of nanovesiclesHeLa cells were grown to sim2 times 107 cells per T175 flaskin 30mL cell culture media in the presence of exosome-depleted FBS From 1mL of this cell medium sim4ndash8 times 109exosomes were isolated with the reagent and sim2ndash4 times 109 withultracentrifugation For the reagent protocol 100 120583Lminus50mLcell media volume inputs was tested and exosome recoverywas linear in this range The process is scalable up ordown depending on the needs for down stream analysismdashforexample for sequencing applications larger amounts of RNAare required compared to qRT-PCR and thus larger scale ofexosome isolation is required from 10ndash50mL cell media

From 100120583L serum sim15ndash3 times 1011 exosomes were recov-ered with the reagent and sim15ndash5 times 1010 with ultracentrifu-gation For the reagent protocol 50 120583Lminus5mL serum volumeinputs was tested and exosome recovery was linear in thisrange For sequencing applications larger amounts of RNAare required compared to qRT-PCR and thus larger scale ofexosome isolation is required from 1ndash5mL serum

Samples were next analyzed by Western blotting withantibodies specific to tetraspanins CD63 and CD9-well char-acterized exosomal markers [11 31] Results are shown inFigures 2(a) and 2(b) (cell media) and Figures 2(c) and2(d) (serum) confirming that CD63- and CD9-positivenanovesicle populations were recovered with both reagent


PGM readsfastq

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998400 adapter-Remove ldquotooshortrdquo reads

Figure 4 Iterative mapping pipeline used to map and quantify RNAs sequenced from both exosomes and parental samples Each read thataligns to a particular RNA is referred to as a ldquocountrdquo of that RNA type Quantitation is accomplished by calculating a tally of the counts foreach type of RNA

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omes

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(b)

Figure 5 RNA sequencing results for exosomes versus parental HeLa cells Stacked bar plots depict mapped read counts distributed by RNAspecies for both HeLa cells and HeLa cell culture media derived exosomes preparations (a) Full scale (b) Zooming into 0ndash10 range on the119910-axis to view less prominent RNA types representation


and ultracentrifugation The above results demonstrate thatboth protocols isolate clean exosome populations but thereagent consistently recovered more exosomes and the pro-tocol is much faster easier and more reliable

32 Isolation of Exosomal RNA Cargo and Analysis by qRT-PCR Once the purity of recovered exosomes was confirmedand the amount determined we proceeded with the isolationand subsequent analysis of the exosomal RNA Using theTotal exosome RNA and protein isolation kit developedspecifically for this purpose samples were put through anorganic extraction followed by immobilization of RNA onglass-fiber filters to purify the total RNA We followed thisprotocol to isolate RNA from exosomes derived from HeLacell culture media and blood serum samples using both theTotal exosome isolation reagents and ultracentrifugation pro-cedure Subsequent analysis with Nanodrop and Bioanalyzerhas shown that for exosomes isolated from 30mL of HeLacell culture media using the ultracentrifugation protocol sim38 ng RNA was recovered The reagent method resulted inisolation of somewhat more exosomes from the same volumeof HeLa cell culture media from which sim75 ng RNA wasextracted For exosomes isolated from 4mL of serum usingthe ultracentrifugation protocol sim09 pg RNA was recoveredThe reagent method resulted in isolation of somewhat moreexosomes from the same volume of serum fromwhich sim2 ngRNA was extracted

Before proceeding with the sequencing analysis of theRNA the levels of five microRNAs miR16 miR24 miR26amiR451 and let7e earlier reported to be present in exosomes[11 21] were quantified by qRT-PCR Results are displayedin Figure 3(a) (cell media) and Figure 3(b) (serum) Based onCt values 25ndash33 for all analytes RNA isolation was efficientand the amount of material recovered is ample for standardPCR analysismdashRNA recovered from exosomes derived from3 120583L serum or 30 120583L cell media is sufficient for one qPCRreaction The reagent method recovered somewhat higherlevels to recover of exosomes compared to ultracentrifugationprocedure as indicated by 1ndash3Ct shift for different RNAsFrom this point we focused on analysis of the RNA contentof exosomes isolated with the reagent

33 Preparation of the Small RNA Libraries and SequencingExosomal RNA Exosomal RNA recovered from cell culturemedia and serum derived samples was analyzed by sequenc-ing and compared to the parental samples whole cells andcell-free serum respectively For the library constructionthe primer binding flanking sequences were ligated to theRNA in the samples followed by reverse transcription andPCR amplification Certainmodifications were introduced tothe standard protocol to address the specific nature of theexosome samples longer ligation and extended amplificationwere performed as relatively low amount of RNA is recoveredfrom the exosomes Once the libraries were constructed theywere sequenced using the Ion Torrent PGM instrument with318 chips following standard protocols

34 Mapping and Counting RNA Sequencing Reads ThePGM 318 chips hold over 11 million wells For the samples

derived from cell media as well as serum 9-10 million wellswere loaded (75ndash90) and sequenced After subtracting thepolyclonals low quality sequences and nontemplated ISPs5-6 million final readouts were obtained from each run Ofthose reads 90ndash98 were mapped

An iterative mapping pipeline was used to map andquantify RNAs sequenced from both exosome and parentalsamples (Figure 4) Each read which aligns to a particularRNA is referred to as a ldquocountrdquo for that RNA type Quanti-tation was accomplished by calculating a tally of counts foreach sample and RNA type

Since no a priori exosome RNA content informationis known for HeLa cell culture media no assumptionswere made about what databases should be referenced formapping quantitation and comparison For this reasonwe designed an iterative mapping strategy for both sam-ple types that included three mapping references utilizedin a specific order This is a ldquocatch allrdquo approach thattakes advantage of published transcriptome annotations andsequences which are publicly available The implementa-tion of this approach involves a very intuitive workflowBriefly reads were first mapped to miRBase [41] microRNAprecursors (hairpins) Any unmapped reads from this stepwere then mapped to NCBI RefSeq [42] transcripts Finallyany remaining unmapped reads at this point were mappedto the Noncodeorg noncoding RNA transcripts whichinclude piwi-interacting (piRNA) small Cajal body-specific(scaRNA) and small nucleolar RNAs (snoRNA)At each stepmapped reads were counted per transcript and printed to afinal report The counts of mapped reads per transcript serveas proxies for representation and can be utlized for relativequantitative comparisons between samples Results fromthe sequencing analysis will be discussed in the followingsections looking at the specific RNA content from exosomesof each sample type

35 Analysis of the Sequencing Data

351 RNA Content of HeLa Cell Culture Media Derived Exo-somes Using mapped sequence read counts we quantifiedthe RNA cargo of exosomes extracted from HeLa cell culturemedia and then compared it to RNA from the ldquoparentalrdquo cellsThis is to our knowledge the first report on RNA contentof exosomes secreted by HeLa cells and also comparingexosomes to the parental samples As illustrated in Figure 5exosomes derived from HeLa cell culture media containedextremely diverse RNA ldquocargordquo As expected from previousresearch a large number of miRNAs and mRNAs weredetected However significant amounts of rRNA tRNA andother RNA types were discovered as well There were cleardifferences in RNA species representation between cells andexosome preparationsThe cell preparation had a high rRNArepresentation about twice the amount in exosomes (sim40)piRNA was present in comparable amounts in both sampletypes while miRNA mRNA RefSeq ncRNA and tRNArepresentation in the exosomal samples was higher than thatof parental cell preparations This observation agrees withthe aforementioned studies in which full length RNA was


0

2

4

6

8

10

12M

appe

d re

ads (

)

miRNA

CellsExosomes

hsa-mir-3665 hsa-mir-4279 hsa-mir-21 hsa-mir-31

(a)

CellsExosomes

0

1

2

3

4

5

6

7

8

MTRNR2L2 LLGL1 SUSD2

Map

ped

read

s (

)

mRNA

(b)

CellsExosomes

0

2

4

6

8

10

12

chr6trna61-MetCAT chr1trna109-GluCTC

Map

ped

read

s (

)

tRNA

(c)

0

2

4

6

8

10

12

14

MALAT1 RNY5 RNY4

Map

ped

read

s (

)

ncRNA

CellsExosomes

(d)

Figure 6 Differential representation of specific RNA in exosomes versus parental HeLa cells miRNAs (a) mRNA (b) tRNA (c) and ncRNA(d) from cells and exosome preparations Quantities were normalized by the percentage of reads aligned to that transcript out of the totalmapped

found to be represented in exosomes and may have a role inintercellular communication

When summarizing total counts per samples type wecalculated mean raw counts per RNA-Seq library and thenranked them from high to low for miRNA tRNA mRNAand ncRNA categoriesThe top ten are listed in Table 1 In thisview representation is not given as a normalized percentagebut as ranks of individual transcripts by category and may be

compared directly between exosomal and parental cell sam-ples For exosomes the transcripts that stand out include hsa-mir-21 hsa-mir-3160-1 chr6trna61-MetCAT chr1trna109-GluCTC SUSD2 mRNA RNY5 ncRNA and RNY4 ncRNAThe biggest overlap between exosomes and parental samplewas observed for tRNA 7 out of 10 top represented sequenceswere the same for miRNA 4 of 10 sequences were the samefor mRNA and ncRNA 2 sequences were the same


0

10

20

30

40

50

60

70

80

90

100

Map

ping

to R

NA

spec

ies (

)

mRNArRNA

tRNAmiRNA

Seru

m d

1 re

p1

Seru

m d

1 re

p2

Seru

m d

2 re

p1

Seru

m d

2 re

p2

Exos

omes

d1

rep1

Exos

omes

d1

rep2

Exos

omes

d2

rep1

Exos

omes

d2

rep2

Figure 7 RNA sequencing results for exosomes versus parentalserum Stacked bar plot depicts mapped read count distribution byRNA species derived from two human blood donors d1 and d2 Tworeplicates (rep1 rep2) were sequenced per donor SnRNA ScaRNASnoRNA piRNA were also detected though at the very low levels

Several targets from four of the different HeLa majorRNA species (miRNA tRNA mRNA and ncRNA) showedsignificant differences in representation between the twosample types (Figure 6) In particularmiR-21miR-31 SUSD2mRNA tRNA61-MetCAT tRNA109-GluCTC RNY5ncRNAand RNY4 ncRNAs were higher in exosomes while miR-3665 miR-4279 MTRNR2L2 mRNA LLGL1 mRNA andMALAT1 ncRNA were significantly higher in parental cellsamplesThe reason for these differences is yet to be exploredand will provide useful information regarding sorting of par-ticular RNA sequences into exosomes Interestingly miR-21has been reported to be a prognostic indicator in several typesof cancer [43ndash45] and both miR-31 and miR-21 have beenreported to be associated with esophageal cancer [46] Thisraises the possibility that a mechanism by which metastasesare induced within a cell population could be via intercellularcommunication by exosomes containing specific miRNAs

It is alsoworth noting fromFigure 6 the level of variability(error bars) seen within replicate libraries prepared fromthree separate HeLa cell cultures Higher count variabilitywas observed for mRNA and ncRNA measurements whencompared to miRNA or tRNA measurements This suggeststhat our libraries contain varying levels of natural degra-dation products and representation of mRNA and ncRNAsfrom exosomes are stochastic in nature Moreover the lowervariability in miRNA representation could be an inference tofunctional importance

352 RNA Content of Human Blood Serum Exosomes Cellculture is a useful model for initial characterization studies

0

2

4

6

8

10

12

14

16

18

Serum1 Serum2 Exosomes1 Exosomes2

Map

ped

read

s (

)

hsa-mir-1281hsa-mir-4257hsa-mir-451

Figure 8 Differential representation of specific miRNAs in exo-somes versus parental serum samples Quantities were normalizedby the percentage of reads aligned to that transcript out of the totalmapped

however blood samples are of higher interest due to theirclinical relevance and potential for use in human diagnosticsIn the latter case more heterogeneity in RNA compositionshould be obviously expected versus cell culture since thesamples are taken frommultiple individualsWe analyzed theRNA cargo of exosomes extracted from the blood serum ofhealthy human donors as well as ldquoparentalrdquo serum samplesAs illustrated in Figure 7 exosomes derived from serumcontain diverse RNA ldquocargordquo miRNA mRNA rRNA tRNAand various ncRNA There were clear differences in RNAspecies representation between whole serum and exosomepreparations Serum preparation had a higher rRNA repre-sentation whereasmRNA tRNA and ncRNA representationin the exosomes was higher than that in serum preparationsmiRNA was present in comparable amounts However whenscrutinizing individualmiRNA representation as a fraction oftotalmapped reads we did observe a set ofmiRNAswhich aredifferentially represented between serum and serum derivedexosome fractions (as discussed later)

WhenRNAcomposition profileswere compared betweenserum and cell culture samples general differences wereobservable Notably rRNA read counts compose a smallerpercentage of total reads in serum while levels of mRNAncRNA make up a higher percentage irrespective of samplereplicate or type In addition the serum and exosomallibraries showed more variability in terms of RNA speciesdistribution in duplicate libraries made from two blooddonors We expect that donor to donor variability may bea challenge in serum samples moving forward so there is aneed to further explore methods to reduce or account for thisvariability for future studies


Table 1 RNA representation in exosomes versus parental HeLa cells top 10 RNA transcripts organized by RNA type Values for cells andexosome preparations represent mean counts of mapped reads to reference transcripts from three replicate cell lysates Accession numbersand gene symbols were obtained from NCBI RefSeq annotation

(a)

miRBase ID Cells miRBase ID Exosomeshsa-mir-4279 73100 hsa-mir-21 201533hsa-mir-1234 50500 hsa-mir-3160-1 100733hsa-mir-451a 41033 hsa-mir-4739 89433hsa-mir-3665 33033 hsa-mir-31 82733hsa-mir-4449 26567 hsa-mir-23a 78367hsa-mir-3960 24067 hsa-mir-24-2 64400hsa-mir-3160-1 23800 hsa-mir-1273a 52167hsa-mir-1273a 23367 hsa-mir-30a 43933hsa-mir-1273g 19533 hsa-mir-451a 37133hsa-mir-92b 18000 hsa-mir-1273g 36800tRNA ID Cells tRNA ID Exosomeschr1trna109-GluCTC 17533 chr6trna61-MetCAT 3412967chr16trna34-GlyCCC 14733 chr1trna109-GluCTC 1592433chr16trna18-GlyGCC 12167 chr16trna18-GlyGCC 569667chr6trna61-MetCAT 10100 chr1trna33-GlyGCC 235433chr17trna28-CysGCA 8633 chr16trna10-LysCTT 211967chr5trna5-ValAAC 5267 chr5trna11-LysCTT 210733chr5trna11-LysCTT 4800 chr13trna3-GluTTC 198700chr1trna33-GlyGCC 3567 chr16trna34-GlyCCC 192833chr17trna10-GlyTCC 3300 chr1trna54-GluCTC 145033chr13trna3-GluTTC 2533 chr1trna124-GlyCCC 136500

(b)

mRNA Accession Cells mRNA Accession ExosomesMTRNR2L2 NM 001190470 479367 SUSD2 NM 019601 317233MTRNR2L8 NM 001190702 264633 BRWD3 NM 153252 50100SETD2 NM 014159 54467 SENP6 NM 015571 36267CUX1 NM 001202543 47533 FAM59B NM 001191033 35533TUBA1C NM 032704 47533 TUBBA4 NM 006087 33833FXR1 NM 004860 36333 QRFPR NM 198179 31333SYT6 NM 001253772 31900 MDK NM 001012334 30700FUS NM 001170634 28733 MTRNR2L2 NM 001190470 30200MDK NM 001012334 23867 CWC25 NM 017748 26600UBE2S NM 014501 23033 DUSP13 NM 001007272 23200ncRNA Accession Cells ncRNA Accession ExosomesSNORA72 NR 002581 386567 RNY5 NR 001571 848267MALAT1 NR 002819 87967 RNY4 NR 004393 620633RNU1-9 NR 004426 79800 RN5S3 NR 023365 234367RN5S1 NR 023363 66867 RNY1 NR 004391 210967RNU2-1 NR 002716 62900 RNY4P8 NR 033356 209433RN7SL1 NR 002715 58167 RNY3 NR 004392 111933SNORD3C NR 006881 53100 RN7SL2 NR 027260 70233VDAC1 NR 036624 43733 SNORD31 NR 002560 61933RN7SK NR 001445 43533 RNU1-5 NR 004400 56167RN7SL2 NR 027260 42567 RN7SL1 NR 002715 45733


Table 2 RNA representation in exosomes versus cell-free parental serum top 10 RNA transcripts organized by RNA type Values for cell-freeserum and exosome preparations represent mean counts of mapped reads to reference transcripts from replicate samples prepared from asingle donor Accession numbers and gene symbols were obtained from NCBI RefSeq annotation

(a)

miRBase ID Serum miRBase ID Exosomeshsa-mir-1246 52225 hsa-mir-1246 21450hsa-mir-451 21635 hsa-mir-451 18370hsa-mir-2115 770 hsa-mir-3934 14445hsa-mir-1281 5310 hsa-mir-1281 10685hsa-mir-486 3375 hsa-mir-3180-1 8710hsa-mir-16-1 3260 hsa-mir-4257 8515hsa-mir-3180-1 1165 hsa-mir-181d 6875hsa-mir-4257 5250 hsa-mir-486 5935hsa-mir-124-1 1360 hsa-mir-766 5805hsa-mir-3167 2935 hsa-mir-497 5650tRNA ID Serum tRNA ID Exosomeschr6trna31-SerGCT 8010 chr6trna4-ArgTCG 20710chr12trna10-AspGTC 7180 chr6trna31-SerGCT 18670chr6trna4-ArgTCG 6615 chr15trna4-ArgTCG 10100chr15trna4-ArgTCG 4750 chr6trna14-TyrGTA 8945chr14trna19-TyrGTA 3645 chr14trna19-TyrGTA 8845chr6trna124-ArgTCG 2375 chr7trna7-CysGCA 6815chr6trna14-TyrGTA 2235 chr1trna105-GlnCTG 5910chr12trna12-AspGTC 2140 chr6trna52-ArgTCT 5810chr6trna145-SerAGA 2110 chr6trna124-ArgTCG 5655chr6trna52-ArgTCT 2005 chr16trna1-ArgCCG 5445

(b)

mRNA Accession Serum mRNA Accession ExosomesCCDC96 NM 153376 2515 CCDC96 NM 153376 6065SIRPG NM 080816 2405 HGSNAT NM 152419 5030TBX22 NM 016954 2150 TBX22 NM 016954 4650RNF223 NM 001205252 2005 SIRPG NM 018556 3500SIRPG NM 018556 1980 SIRPG NM 080816 3400NFIX NM 002501 1980 NFIX NM 002501 3385RAP2A NM 021033 1740 GPR126 NM 198569 2880SIRPG NM 001039508 1540 ILDR2 NM 199351 2735HGSNAT NM 152419 1430 PAPOLB NM 020144 2675RBFOX1 NM 001142333 1295 SIRPG NM 001039508 2465ncRNA Accession Serum ncRNA Accession ExosomesRNY4 NR 004393 10435 RNY4 NR 004393 7045RNY5 NR 001571 4770 RNY5 NR 001571 2620RNY3 NR 004392 3600 RNY3 NR 004392 1740RNY4P8 NR 033356 1770 FLJ33534 NR 040080 1670RNU2-1 NR 002716 1595 LOC100130673 NR 038454 1550RNY1 NR 004391 1455 LOC100507605 NR 038375 1530RNU12 NR 029422 1275 C20orf173 NR 026933 1520C1orf213 NR 033690 1010 LOC100507605 NR 038374 1395RNU5B-1 NR 002757 710 LOC644936 NR 004845 1360C20orf173 NR 026933 705 LOC100507605 NR 038376 1305


Table 2 shows the representation of top 10 targetsfrom each RNA species present in serum miRNA tRNAmRNA and ncRNA For exosomes the sequences that standout include hsa-mir-1246 hsa-mir-451 chr6trna4-ArgTCGchr6trna31-SerGCT CCDC96 mRNA HGSNAT mRNARNY4 ncRNA and RNY5 ncRNA The highest overlapbetween exosomes and parental serum was observed fortRNA andmRNA 7 out of 10 top represented sequences werethe same in both cases for miRNA 6 sequences out of 10were the same for ncRNA 4 sequences were the same Exceptfor ncRNA there was little overlap betweenmost representedsequences in serum derived exosomes and HeLa cell derivedexosomes

Levels of certain RNA sequences were substantially dif-ferent between exosomes and parental serum sample asillustrated in Figure 8 A subset of 3 miRNA targets showeddifferent representation profiles between the two sampletypes miR-1281 and miR-4257 were higher in exosomeswhile miR-451 was significantly higher in parental samplemiR-451 for instance was reported as associated with resis-tance to chemotherapy in ovarian cancer [47] as well asplaying a part in controlling glioma cell proliferation andmigration [48] In additional it has also been reported aspotential marker for renal cell carcinoma with much lowerlevels in RCC patients [49] This miRNA has a number ofimportant intracellular functions but potenially no value forcell signaling purposes and thus its levels within exosomesare low The above miRNAs could potentially be utilized asexosomal markers as currently available options are limitedto a handful of proteins including CD63 CD81 and CD9none of which seems to be absolutely specific to exosomes

The mechanism of sorting particular RNA sequencesinto exosomes is not well understood at the moment andis extremely challenging to study Each cell secretes severaltypes of exosomes andmicrovesicles with different functionsand containing different cargo For example some of thesevesicles simply allow cells to expel unnecessary RNA andproteins while others allow cells to send signaling or affectormolecules to other cell populations within the body [6]When analyzing exosomes from body fluids the situationbecomes increasingly complicated as many cell types con-tribute exosomes to the population Vitally needed is thedevelopment of tools allowing the study of the mechanismsof formation of the exosomes their secretion trafficking andinternalization in the recipient cells It is also crucial to enableseparation of various classes or subtypes of the exosomesbased on their surface protein signature or other parameters

4 ConclusionsWe have successfully developed a set of reagents and acomplete exosome workflow solution starting from fast andefficient extraction of exosomes from cell culture media andblood serum to robust isolation of RNA and characteriza-tion of exosomal RNA content using qRT-PCR and next-generation sequencing methodsThe procedure is completedin a fraction of the time compared to the current standardprotocols utilizing ultracentrifugation and allows recoveryof fully intact exosomes in higher yields Analysis of the

sequencing data revealed a very sophisticated RNA contentin exosomes with most of the cellular coding and noncodingRNA present in exosomes but in many cases at significantlydifferent levels and ratios It will be of paramount importanceto gain a better understanding regarding the sorting ofparticular RNA sequences into exosomes and further intodifferent exosome subpopulations The work described hereis the first step towards developing standardized techniquesand protocols for isolation of exosomes and downstreamanalysis of their constituents These reagents and workflowswill be highly useful for scientists working on both basicresearch aspects of exosomes and developing next-generationminimally invasive diagnostics

Conflict of InterestsJeoffrey Schageman Emily Zeringer Mu Li Tim Barta KristiLea Jian Gu Susan Magdaleno Robert Setterquist andAlexander V Vlassov are employees of Life Technologies andTotal exosome isolation reagent Total exosome RNA andprotein isolation kit as well as certain reagents and instru-ments for qPCR and sequencing analysis are manufacturedby Invitrogen Ion Torrent and Applied Biosystems parts ofLife Technologies

Authorsrsquo Contribution

Jeoffrey Schageman and Emily Zeringer contributed equallyto this paper

References

[1] J S Schorey and S Bhatnagar ldquoExosome function from tumorimmunology to pathogen biologyrdquo Traffic vol 9 no 6 pp 871ndash881 2008

[2] M Mittelbrunn and F Sanchez-Madrid ldquoIntercellular commu-nication diverse structures for exchange of genetic informa-tionrdquo Nature Reviews Molecular Cell Biology vol 13 no 5 pp328ndash335 2012

[3] G van Niel I Porto-Carreiro S Simoes and G RaposoldquoExosomes a common pathway for a specialized functionrdquoJournal of Biochemistry vol 140 no 1 pp 13ndash21 2006

[4] A Lakkaraju and E Rodriguez-Boulan ldquoItinerant exosomesemerging roles in cell and tissue polarityrdquoTrends in Cell Biologyvol 18 no 5 pp 199ndash209 2008

[5] D Xu and H Tahara ldquoThe role of exosomes and microRNAsin senescence and agingrdquo Advanced Drug Delivery Reviews vol65 no 3 pp 368ndash375 2013

[6] C Thery L Zitvogel and S Amigorena ldquoExosomes composi-tion biogenesis and functionrdquoNature Reviews Immunology vol2 no 8 pp 569ndash579 2002

[7] A V Vlassov S Magdaleno R Setterquist and R ConradldquoExosomes current knowledge of their composition biologicalfunctions and diagnostic and therapeutic potentialsrdquo Biochim-ica et Biophysica Acta vol 1820 no 7 pp 940ndash948 2012

[8] L Balaj R Lessard L Dai et al ldquoTumourmicrovesicles containretrotransposon elements and amplified oncogene sequencesrdquoNature Communications vol 2 no 1 Article ID ncomms1180pp 180ndash185 2011


[9] A Janowska-Wieczorek M Wysoczynski J Kijowski et alldquoMicrovesicles derived from activated platelets induce metas-tasis and angiogenesis in lung cancerrdquo International Journal ofCancer vol 113 no 5 pp 752ndash760 2005

[10] P W Kriebel V A Barr E C Rericha G Zhang and C AParent ldquoCollective cell migration requires vesicular traffickingfor chemoattractant delivery at the trailing edgerdquo Journal of CellBiology vol 183 no 5 pp 949ndash961 2008

[11] H Valadi K Ekstrom A Bossios M Sjostrand J J Leeand J O Lotvall ldquoExosome-mediated transfer of mRNAs andmicroRNAs is a novel mechanism of genetic exchange betweencellsrdquo Nature Cell Biology vol 9 no 6 pp 654ndash659 2007

[12] J Skog T Wurdinger S van Rijn et al ldquoGlioblastomamicrovesicles transport RNAand proteins that promote tumourgrowth and provide diagnostic biomarkersrdquoNature Cell Biologyvol 10 no 12 pp 1470ndash1476 2008

[13] K Al-Nedawi B Meehan J Micallef et al ldquoIntercellulartransfer of the oncogenic receptor EGFRvIII by microvesiclesderived from tumour cellsrdquo Nature Cell Biology vol 10 no 5pp 619ndash624 2008

[14] L J Vella R A Sharples V A Lawson C LMasters R Cappaiand A F Hill ldquoPackaging of prions into exosomes is associatedwith a novel pathway of PrP processingrdquo Journal of Pathologyvol 211 no 5 pp 582ndash590 2007

[15] S A Bellingham B M Coleman and A F Hill ldquoSmall RNAdeep sequencing reveals a distinct miRNA signature releasedin exosomes from prion-infected neuronal cellsrdquo Nucleic AcidsResearch 2012

[16] D M Pegtel K Cosmopoulos D A Thorley-Lawson et alldquoFunctional delivery of viral miRNAs via exosomesrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 107 no 14 pp 6328ndash6333 2010

[17] P Leblanc S Alais I Porto-Carreiro et al ldquoRetrovirus infectionstrongly enhances scrapie infectivity release in cell culturerdquoEMBO Journal vol 25 no 12 pp 2674ndash2685 2006

[18] N Spielmann and D T Wong ldquoSaliva diagnostics and thera-peutic perspectivesrdquo Oral Diseases vol 17 no 4 pp 345ndash3542011

[19] J Nilsson J Skog A Nordstrand et al ldquoProstate cancer-derivedurine exosomes a novel approach to biomarkers for prostatecancerrdquoThe British Journal of Cancer vol 100 no 10 pp 1603ndash1607 2009

[20] P S Mitchell R K Parkin E M Kroh et al ldquoCirculatingmicroRNAs as stable blood-based markers for cancer detec-tionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 30 pp 10513ndash10518 2008

[21] S Mathivanan C J Fahner G E Reid and R J SimpsonldquoExoCarta 2012 database of exosomal proteins RNA andlipidsrdquo Nucleic Acids Research vol 40 pp D1241ndashD1244 2012

[22] P Diehl A Fricke L Sander et al ldquoMicroparticles majortransport vehicles for distinct microRNAs in circulationrdquo Car-diovascular Research vol 93 no 4 pp 633ndash644 2012

[23] E N Nolte-rsquot Hoen H P Buermans M Waasdorp W Stoor-vogel M H Wauben and P A T Hoen ldquoDeep sequencing ofRNA from immune cell-derived vesicles uncovers the selectiveincorporation of small non-codingRNAbiotypeswith potentialregulatory functionsrdquoNucleic Acids Research vol 40 no 18 pp9272ndash9285 2012

[24] W Koh C T Sheng B Tan et al ldquoAnalysis of deep sequencingmicroRNA expression profile from human embryonic stem

cells derivedmesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of hepatic nuclearfactor 4 alphardquo BMC Genomics vol 11 no 1 article S6 2010

[25] S S Luo O Ishibashi G Ishikawa et al ldquoHuman villous tro-phoblasts express and secrete placenta-specificmicroRNAs intomaternal circulation via exosomesrdquo Biology of Reproductionvol 81 no 4 pp 717ndash729 2009

[26] Q Zhou M Li X Wang et al ldquoImmune-related microRNAsare abundant in breast milk exosomesrdquo International Journal ofBiological Sciences vol 8 no 1 pp 118ndash123 2011

[27] M Mittelbrunn C Gutierrez-Vazquez C Villarroya-Beltri etal ldquoUnidirectional transfer of microRNA-loaded exosomesfrom T cells to antigen-presenting cellsrdquo Nature Communica-tions vol 2 no 1 article 282 2011

[28] A Zomer T Vendrig E S Hopmans M van Eijndhoven J MMiddeldorp and D M Pegtel ldquoExosomes fit to deliver smallRNArdquo Communicative and Integrative Biology no 5 pp 447ndash450 2010

[29] G Rabinowits C Gercel-Taylor J M Day D D Taylor andG H Kloecker ldquoExosomal microRNA a diagnostic marker forlung cancerrdquoClinical LungCancer vol 10 no 1 pp 42ndash46 2009

[30] D D Taylor and C Gercel-Taylor ldquoMicroRNA signatures oftumor-derived exosomes as diagnostic biomarkers of ovariancancerrdquo Gynecologic Oncology vol 110 no 1 pp 13ndash21 2008

[31] C Thery S Amigorena G Raposo and A Clayton ldquoIsolationand characterization of exosomes fromcell culture supernatantsand biological fluidsrdquo Current Protocols in Cell Biology vol 3 p322 2006

[32] httpwwwsystembiocom[33] httpwwwhansabiomedeu[34] httpwwwbiooscientificcom[35] K Wang S Zhang J Weber D Baxter and D J Galas

ldquoExport of microRNAs and microRNA-protective protein bymammalian cellsrdquo Nucleic Acids Research vol 38 no 20 pp7248ndash7259 2010

[36] B Ewing L Hillier M C Wendl and P Green ldquoBase-calling of automated sequencer traces using phred I Accuracyassessmentrdquo Genome Research vol 8 no 3 pp 175ndash185 1998

[37] M Martin ldquoCutadapt removes adapter sequences from high-throughput sequencing readsrdquo EMBnet Journal vol 17 no 1article 1 2012

[38] M David M Dzamba D Lister L Ilie and M BrudnoldquoSHRiMP2 sensitive yet practical short read mappingrdquo Bioin-formatics vol 27 no 7 pp 1011ndash1012 2011

[39] M Alvarez M Khosroheidari R Kanchi Ravi and J DistefanoldquoComparison of protein microRNA and mRNA yields usingdifferent methods of urinary exosome isolation for the discov-ery of kidney disease biomarkersrdquo Kidney International vol 82no 9 pp 1024ndash1032 2012

[40] S He C Liu G Skogerboslash et al ldquoNONCODE v20 decodingthe non-codingrdquoNucleic Acids Research vol 36 no 1 pp D170ndashD172 2008

[41] A Kozomara and S Griffiths-Jones ldquoMiRBase integratingmicroRNA annotation and deep-sequencing datardquo NucleicAcids Research vol 39 no 1 pp D152ndashD157 2011

[42] K Pruitt T Tatusova G Brown and D Maglott ldquoNCBIreference sequences (RefSeq) current status new features andgenome annotation policyrdquo Nucleic Acids Research vol 40 ppD130ndashD135 2012

[43] H Shibuya H Iinuma R Shimada A Horiuchi and TWatan-abe ldquoClinicopathological and prognostic value ofmicroRNA-21


and microRNA-155 in colorectal cancerrdquo Oncology vol 79 no3-4 pp 313ndash320 2011

[44] I G Papaconstantinou AMantaMGazouli et al ldquoExpressionof microRNAs in patients with pancreatic cancer and itsprognostic significancerdquo Pancreas vol 42 no 1 pp 67ndash71 2013

[45] H Faragalla YM Youssef A Scorilas et al ldquoThe clinical utilityof miR-21 as a diagnostic and prognostic marker for renal cellcarcinomardquo Journal of Molecular Diagnostics vol 14 no 4 pp385ndash392 2012

[46] W O Lui N Pourmand B K Patterson and A Fire ldquoPatternsof known and novel small RNAs in human cervical cancerrdquoCancer Research vol 67 no 13 pp 6031ndash6043 2007

[47] M T M van Jaarsveld J Helleman E M J J Berns andE A C Wiemer ldquoMicroRNAs in ovarian cancer biology andtherapy resistancerdquo International Journal of Biochemistry andCell Biology vol 42 no 8 pp 1282ndash1290 2010

[48] J Godlewski A Bronisz M O Nowicki E A Chiocca andS Lawler ldquomicroRNA-451 a conditional switch controllingglioma cell proliferation and migrationrdquo Cell Cycle vol 9 no14 pp 2742ndash2748 2010

[49] M Redova A Poprach J Nekvindova et al ldquoCirculating miR-378 and miR-451 in serum are potential biomarkers for renalcell carcinomardquo Journal of Translational Medicine vol 10 no 1article 55 2012


cells [24] placenta [25] and breast milk [26] Except forNolte-rsquot Hoen et al [23] who characterized awide populationof small RNA residing within the vesicles all studies solelyfocus on analysis of the miRNA content Significantly lessinformation is available regarding other RNA classes andother types of body fluids such as blood Moreover certainreports contain a number of contradictions Several papersfor example indicate that the RNA ldquocargordquo of exosomes issubstantially different from the parental cell content [12 2728] This runs counter to several other authors working withcancer cells who have noted that themiRNA content for theiroriginating cancer cells is similar to that found in circulatingexosomes opening up the possibility that exosomes could beused as unique diagnostic markers [29 30]The complicatingfactor between these studies is a lack of standardized tech-niques protocols and workflows for isolation of exosomesand downstream analysis of their constituents Currently themost popular approach for isolation of exosomes is basedon ultracentrifugation [31] which allows the researcher toobtain highly pure exosomes however it is a very lengthydifficult and unreliable process To address the urgent needto have a simple solution for isolation of exosomes severalreagents were developed by biotechnology companies in thelast two years System Biosciences released a proprietaryreagent named ExoQuick that can be added to serumconditioned cell media or urine and is claimed to precipitatethe exosomes [32] HansaBioMed is offering an array ofproducts called ExoTest kitsmdashfeaturing anti-CD63 -CD81or -CD9 antibodies immobilized on 96-well platesmdashforexosome capturing and characterization [33] Bioo Scientificlaunched the ExoMir kit that essentially removes all cellsplatelets and cellular debris on one microfilter and capturesall vesicles bigger than 30 nm on a second microfilter usingpositive pressure to drive the fluid [34] One should keep inmind that depending on the isolation procedure exosomepreparations are contaminated to a various extent with othermicrovesicles or RNA-protein complexes [35] dramaticallyaffecting the outcome of downstream analysis

Here we describe a complete exosome workflow solutionstarting from fast and efficient extraction of exosomes fromcell culture media and serum to robust isolation of RNA andcharacterization of exosomal RNA content using qRT-PCRand next-generation sequencing techniques Effectivenessof this workflow is demonstrated by analysis of the RNAcontent of exosomes derived from HeLa cells and bloodserum using Ion Torrent PGM as a sequencing platformThis straightforward standardized workflow can be utilizedfor analysis of the exosomal RNA cargo shedding somelight on the mechanisms of formation preferential loadingand functions of these fascinating vesicles In a similarfashion disease-specific RNA signatures residing withinthe exosomes can be uncovered and potentially used fordevelopment of minimally invasive or noninvasive diag-nostics providing the possibility of someday being able toanalyze RNA ldquocontentrdquo of organs and tissues without biopsy

2 Materials and Methods21 Materials Total exosome isolation (from serum) reagent(Invitrogen) Total exosome isolation (from cell culturemedia) reagent (Invitrogen) Total exosome RNA and proteinisolation kit (Invitrogen) blood serum from two donorscell culture media from HeLa cells 10x PBS nuclease-freewater (Ambion) 100 ethanol nonoptical adhesive cov-ers (Applied Biosystems) optical adhesive covers (AppliedBiosystems) 384-well PCR standard plates (Applied Biosys-tems) 96-well PCR standard plates (Applied Biosystems)universal PCR master mix II (Applied Biosystems) humanTaqMan miRNA assays Veriti 96-well thermocyclers (Ap-plied Biosystems) 7900HT Instrument SW v23 TaqManmicroRNA reverse transcription kit (Applied Biosystems)1000 reactions and Ion Total RNA-Seq kit v2 (Life Technolo-gies) were utilized

22 Extraction of Exosomes from Serum and Cell Media UsingTotal Exosome Isolation Reagents

221 Cell Culture Media Fresh cell media was harvestedfrom HeLa cells grown in T175 flasks Initially the cells weregrown in media containing 10 FBS (to sim90 cell density)then washed twice with PBS and grown for the remaining12 h in 10 exosome-depleted FBS The cell media sampleswere then centrifuged at 2000 g for 30min to remove celldebris The supernatant containing the cell-free cell mediawas transferred to a fresh container and held on ice until useNext each sample was combined with 12 volume of Totalexosome isolation (from cell media) reagent and mixed wellby vortexing or pipetting up and down until a homogenoussolution was formed Typical cell media volume utilizedwas 1mL however the range of 100 120583Lndash50mL was useddepending on the downstream applicationThe samples wereincubated at 4∘C overnight and then centrifuged at 4∘C at10000 g for 1 hThe supernatant was aspirated and discardedand the exosome pellet was resuspended in PBS buffer andthen stored at 4∘C short term (1ndash7 days) or minus20∘C long term

222 Human Blood Serum Frozen serum samples werethawed in a water bath at room temperature until sampleswere completely liquid and then centrifuged at 2000 gfor 30min to remove any cellular debris The supernatantcontaining the cell-free serum was transferred to a freshcontainer and briefly held on ice until use Next each serumsample was combined with 15th volume of Total exosomeisolation (from serum) reagent and then mixed well byvortexing or pipetting up and down until a homogenous solu-tion was formed Typical serum volume utilized was 100 120583Lhowever the range of 50 120583Lndash5mL was used depending onthe downstream application The samples were incubated at4∘C for 30min and then centrifuged at room temperatureat 10000 g for 10min The supernatant was aspirated anddiscarded and the exosome pellet was resuspended in PBSbuffer and then stored at 4∘C short term (1ndash7 days) or minus20∘Cfor long term





















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(a)

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Ultracentrifugation

10 30 50 70 90 110

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(d)








Reag

ent

CD63

Ultr

acen

trifu

gatio

n

(a)

CD9

Reag

ent

Ultr

acen

trifu

gatio

n

(b)

CD63

Reag

ent

Ultr

acen

trifu

gatio

n

(c)

CD9

Reag

ent

Ultr

acen

trifu

gatio

n

(d)


000

500

1000

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3500

4000

hsa-

miR

16

hsa-

miR

24

hsa-

miR

26a

hsa-

miR

451

hsa-

Let7

e

Aver

age C

t val

ues


(a)


000

500

1000

1500

2000

2500

3000

3500

4000

hsa-

miR

16

hsa-

miR

24

hsa-

miR

26a

hsa-

miR

451

hsa-

Let7

e

Aver

age C

t val

ues

(b)






PGM readsfastq

Trimmed fastq





SAM files



step


sample comparisons

Raw sequence

Preprocess

Mapping

Quantify



1∘mapping 3





0

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ping

to R

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)



Cel

ls1

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ls3

Exos

omes

1

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omes

2

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omes

3

(a)

0

1

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omes

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omes

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(b)














0

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(a)

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0

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8


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s (

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(d)






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m d

2 re

p1

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m d

2 re

p2

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omes

d1

rep1

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omes

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(a)

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10 30 50 70 90 110

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Particle size (nm)

(b)

020406080

100120140160180

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10 30 50 70 90 110

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(c)

020406080

100120140160180

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6m

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Ultracentrifugation

10 30 50 70 90 110

130

150

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Particle size (nm)

(d)








Reag

ent

CD63

Ultr

acen

trifu

gatio

n

(a)

CD9

Reag

ent

Ultr

acen

trifu

gatio

n

(b)

CD63

Reag

ent

Ultr

acen

trifu

gatio

n

(c)

CD9

Reag

ent

Ultr

acen

trifu

gatio

n

(d)


000

500

1000

1500

2000

2500

3000

3500

4000

hsa-

miR

16

hsa-

miR

24

hsa-

miR

26a

hsa-

miR

451

hsa-

Let7

e

Aver

age C

t val

ues


(a)


000

500

1000

1500

2000

2500

3000

3500

4000

hsa-

miR

16

hsa-

miR

24

hsa-

miR

26a

hsa-

miR

451

hsa-

Let7

e

Aver

age C

t val

ues

(b)






PGM readsfastq

Trimmed fastq





SAM files



step


sample comparisons

Raw sequence

Preprocess

Mapping

Quantify



1∘mapping 3





0

10

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ping

to R

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Cel

ls1

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ls3

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omes

1

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2

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omes

3

(a)

0

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m d

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p1

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m d

2 re

p2

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omes

d1

rep1

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omes

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rep2

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rep1

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rep2





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acen

trifu

gatio

n

(a)

CD9

Reag

ent

Ultr

acen

trifu

gatio

n

(b)

CD63

Reag

ent

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acen

trifu

gatio

n

(c)

CD9

Reag

ent

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acen

trifu

gatio

n

(d)


000

500

1000

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2000

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3500

4000

hsa-

miR

16

hsa-

miR

24

hsa-

miR

26a

hsa-

miR

451

hsa-

Let7

e

Aver

age C

t val

ues


(a)


000

500

1000

1500

2000

2500

3000

3500

4000

hsa-

miR

16

hsa-

miR

24

hsa-

miR

26a

hsa-

miR

451

hsa-

Let7

e

Aver

age C

t val

ues

(b)






PGM readsfastq

Trimmed fastq





SAM files



step


sample comparisons

Raw sequence

Preprocess

Mapping

Quantify



1∘mapping 3





0

10

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ping

to R

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ies (

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ls1

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ls3

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3

(a)

0

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omes

3

(b)














0

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CellsExosomes


(a)

CellsExosomes

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m d

1 re

p1

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m d

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p2

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m d

2 re

p1

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m d

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omes

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rep2

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rep2





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s (

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(a)


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PGM readsfastq

Trimmed fastq





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step


sample comparisons

Raw sequence

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Mapping

Quantify



1∘mapping 3





0

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ping

to R

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ls3

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omes

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omes

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(a)

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(a)

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(c)

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CellsExosomes

(d)






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ping

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spec

ies (

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mRNArRNA

tRNAmiRNA

Seru

m d

1 re

p1

Seru

m d

1 re

p2

Seru

m d

2 re

p1

Seru

m d

2 re

p2

Exos

omes

d1

rep1

Exos

omes

d1

rep2

Exos

omes

d2

rep1

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omes

d2

rep2





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(a)

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(b)

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tRNA

(c)

0

2

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s (

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CellsExosomes

(d)






0

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ping

to R

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spec

ies (

)

mRNArRNA

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Seru

m d

1 re

p1

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m d

1 re

p2

Seru

m d

2 re

p1

Seru

m d

2 re

p2

Exos

omes

d1

rep1

Exos

omes

d1

rep2

Exos

omes

d2

rep1

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omes

d2

rep2





0

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0

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p1

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m d

2 re

p2

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omes

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omes

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the complete exosome workflow solution: from isolation to … · 2016. 5. 14. · 10 30 50 70 90...

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