One Step Forward, Two Steps Back; Xeno-MicroRNAs Reported in Breast Milk Are Artifacts

17 Pages·2016·0.68 MB·English

RESEARCHARTICLE
One Step Forward, Two Steps Back; Xeno-
MicroRNAs Reported in Breast Milk Are
Artifacts
CanerBağcı1,JensAllmer2,3*
1 DepartmentofBiotechnology,IzmirInstituteofTechnology,Urla,Izmir,Turkey,2 DepartmentofMolecular
BiologyandGenetics,IzmirInstituteofTechnology,Urla,Izmir,Turkey,3 BioniaIncorporated,IZTEKGEB
A8,Urla,Izmir,Turkey
*[email protected]
Abstract
Background
OPENACCESS
MicroRNAs(miRNAs)areshortRNAsequencesthatguidepost-transcriptionalregulation
Citation:BağcıC,AllmerJ(2016)OneStep
ofgeneexpressionviacomplementaritytotheirtargetmRNAs.Discoveredonlyrecently,
Forward,TwoStepsBack;Xeno-MicroRNAs
ReportedinBreastMilkAreArtifacts.PLoSONE11 miRNAshavedrawnalotofattention.Multipleproteincomplexesinteracttofirstcleavea
(1):e0145065.doi:10.1371/journal.pone.0145065 hairpinfromnascentRNA,exportitintothecytosol,trimitsloop,andincorporateitintothe
Editor:VinodScaria,CSIRInstituteofGenomicsand RISCcomplexwhichisimportantforbindingitstargetmRNA.Thisprocessworkswithin
IntegrativeBiology,INDIA onecell,butcirculatingmiRNAshavebeendescribedsuggestingaroleincell-cell
Received:February4,2015 communication.
Accepted:November25,2015
Motivation
Published:January29,2016
VirusesandintracellularparasiteslikeToxoplasmagondiiusemiRNAstomanipulatehost
Copyright:©2016Bağcı,Allmer.Thisisanopen
geneexpressionfromwithinthecellularenvironment.However,recentresearchhas
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits claimedthataricemiRNAmayregulatehumangeneexpression.Despiteongoingdebates
unrestricteduse,distribution,andreproductioninany aboutthesefindingsandgeneralreluctancetoacceptthem,arecentreportclaimedthat
medium,providedtheoriginalauthorandsourceare
foodborneplantmiRNAspassthroughthedigestivetract,travelthroughbloodtobeincor-
credited.
poratedbyalveolarcellsexcretingmilk.ThemiRNAsarethensaidtohavesomeimmune-
DataAvailabilityStatement:Allrawdatausedis
relatedfunctioninthenewborn.
alreadypubliclyavailableandsuitableaccession
numbersareprovidedinthemanuscript.Allrelevant
PrincipalFindings
datafromtheanalysisisprovidedassupplementary
tables.
Weacquiredthedatathatsupportstheirclaimandperformedfurtheranalyses.Inaddition
Funding:ThisstudywassupportedbyagranttoJA tothereportedmiRNAs,wewereabletodetectalmostcompletemRNAsandfoundthatthe
fromtheTheScientificandTechnologicalResearch
foreignRNAexpressionprofilesamongsamplesareexceedinglysimilar.Inspectingthe
CouncilofTurkey(TUBITAK;http://www.tubitak.gov.
sourceofthedatahelpedunderstandhowRNAscouldcontaminatethesamples.
tr/en)withthegrantnumber113E326.Thefunders
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe Conclusion
manuscript.BioniaIncorporatedprovidedsupportin
ViewingthesefindingsincontextwiththedifficultiesforeignRNAsfaceontheirrouteinto
theformofcomputationalresourcesforauthorJA,
butdidnothaveanyadditionalroleinthestudy breastmilkandthefactthatmanyidentifiedfoodbornemiRNAsarenotfromactualfood
PLOSONE|DOI:10.1371/journal.pone.0145065 January29,2016 1/17
Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
design,datacollectionandanalysis,decisionto sources,wecanconcludebeyondreasonabledoubtthattheoriginalclaimsandevidence
publish,orpreparationofthemanuscript.Thespecific presentedmaybeduetoartifacts.Wereportthatthestudyclaimingtheirexistenceismore
roleofthisauthorisarticulatedinthe'author
likelytohavedetectedRNAcontaminationthanmiRNAs.
contributions'section.
CompetingInterests:Theauthorshavethe
followinginterests:JensAllmerisemployedbyBionia
Incorporated.Therearenopatents,productsin
developmentormarketedproductstodeclare.This
doesnotalteradherencetoallthePLOSONE
Introduction
policiesonsharingdataandmaterials,asdetailed
onlineintheguideforauthors. MaturemicroRNAs(miRNAs)areshortRNAs(~22nt)thatguidepost-transcriptionalgene
regulationbybasepairingwiththeirtargetmRNAs.TheywerefirstdiscoveredinCaenorhab-
ditiselegans[1]andhavesinceattractedincreasingattention.MicroRNAshavebeenfoundin
speciesrangingfromvirusestohumans[2,3].MaturemiRNAsderivefromastemloopstruc-
turecalledpre-miRNAwhich,inturn,iscleavedfromapri-miRNAbyDrosha(metazoan;
DCL1forplants).ThematuremiRNAisproducedbyDicerandRISCwhichincorporatesone
sequenceintoitscomplextoguiderecognitionoftargetmRNAs.Moreinformationaboutthis
processcanbefoundinrecentreviews[4,5].MicroRNAsareimportantregulatorsofgene
expressionandtheirdysregulationmayleadtodisease[6].Ithasbeenestablishedthatviruses
encodemiRNAsthatcanregulatehostgeneexpression[7].Itisobviouslyadvantageousforthe
virustocontroltheexpressionofalargearrayofgenesbyencodingforasmallnumberofmiR-
NAs.Ontheotherhand,thehostmayalsoencodeformiRNAsthatcantargetvirusmRNAs;
orcanlosetargetsforthevirus-encodedmiRNAsduringevolution[8].ForeignmiRNAs,
whichwewillcallxeno-miRNAsinthefollowingtext,thus,couldpotentiallycausecross-king-
domgeneregulation.Forvirusesthisregulationoptionseemsevidentandwerecentlyper-
formedastudywhichshowsthatintracellularpathogenslikeToxoplasmagondii,mayalso
employthistypeofregulation[9].Accordingtoourcomputationalanalysis,T.gondiimaybe
abletosecretexeno-miRNAsintoitshostcelltoregulategeneexpression.Virusesandcell
invasivepathogenslikeT.gondiicandirectlyinteractwiththegeneexpressionoftheirhost.
ForfoodbornemiRNAswhichwerefirstproposedbyZhangetal.[10]suchdirectinteraction
is,however,notpossible.Inthiscase,themiRNAhastotoleratefoodprocessingsteps,pass
throughthedigestivetractandthegastrointestinalbarrierintothebloodbeforeitcanreach
thecellstoregulategeneexpression.Thisfindinghasbeencontestedmultipletimesandmost
recentlybyRWLusk[11].RWLuskwasnotabletosecuretheactualmeasurementsbyZhang
etal.,butshowedexperimentally,thatfindingafoodbornemiRNAinplasmaishighlyunlikely.
AroundthesametimeasRWLuskpublishedhisfindings,LukasikandZielenkiewiczreported
thefindingoffoodbornemiRNAsinhumanandporcinebreastmilk[12].Theauthorstook
inspirationfromthefindingsofZhangetal.andanalyzedpubliclyavailablehumanandpor-
cinebreastmilksamples[13,14]whichwereanalyzedusingnextgenerationsequencing(NGS)
foradifferentpurposeattheSichuanAgriculturalUniversity,Sichuan,China.Theauthors
ignoreanycontestofthepaperbyZhangetal.andreportfindinglargeamountsoffoodborne
miRNAsfrommultipleplantspecies;abundantlyamongthemArabidopsisthalianamiRNAs.
SinceArabidopsisisnotafoodsource,thisprovocativefindinginspiredthepresentstudy,
contestingthefindingsofLukasikandZielenkiewicz.Wesuccessfullyrepeatedtheiranalyses
butthenwentfurtherandshowedthatnotonlymiRNAsbutalsolongertranscriptscanbe
foundinthesamples.Furthermore,thesetoftranscriptssharedamongsamples(intra-and
inter-species)washighlycorrelated.Thechanceforsuchhighcorrelationtooccurisextremely
lowand,therefore,webelieveitcanmucheasierbeexplainedthroughcontaminationduring
samplepreparation.Thisnotionisfurthersupportedbythefindingthatatthesametimewhen
thesamplesweremeasuredattheSichuanAgriculturalUniversityallspeciesreportedlyfound
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
tocontributetothemiRNAsinbreastmilkwereanalyzedatthesameinstitute.Intriguingly,
thespecieswithhighernumberofpublicationsalsotendtohavehigheramountofcontamina-
tionintheanalyzedsamples,furthersupportingthisclaim.
Themostlikelyconclusionwecandrawfromouranalysisisthatthesamplesmeasuredat
theSichuanAgriculturalUniversitywerecontaminatedduringtheexperimentalprocedure,
butaftersampling.Thisfinding,inturn,meansLukasikandZielenkiewiczfoundRNAs,but
thattheirconclusionthattheywerexeno-miRNAsperformingcross-kingdomregulationwas
notwellsupportedintheexperimentalevidence.
ResultsandDiscussion
ManyofthePreviouslyReportedPlantsarenotFoodSources
ManymiRNAsreportedbyLukasikandZielenkiewicz[12]tobepresentinbreastmilkorigi-
natefromArabidopsisthalianaanditrankedhighamongthespeciesfoundintheirstudy
(Table1).SinceA.thalianaisnotafoodsource,wewantedtoassesswhethertheotherspecies
thatwerereportedare(Table1).Table1wascreatedbysummarizingthesupportinginforma-
tionprovidedbyLukasikandZielenkiewicz,basicallylistingtheplantspeciesthattheyidenti-
fiedwiththeirsignificantidentificationsforhumanandpigindicatedinthenotecolumn
(Table1).Wefurtheradded6animalspeciesaseitherfoodsourcesornegativecontrols.
Excludingouranimaladditions,29plantspecies’miRNAswerefound9ofwhichwerenot
foodsourcesandanother7whichwereunlikely.70%oftheplantslistedareedible(notneces-
sarilystaplefoods,though),butatleastoneispoisonousandanotheriscoatedtopreventbeing
eaten.Poplartreebarkforexamplemaybepreparedasteaandusedforitssalicincontent,but
wedoubtthatinthismanneranydetectableamountofRNAwillbefoundinhumansamples.
Usingaconservativeapproach,60%ofthespecies,whosemiRNAswerereportedlydetectedin
breastmilk(Table1),arenotfoodsourcesforhumanorpig.
Otherfoodsourcesaremissingfromthetable,especiallyforSichuanwheretheexperiments
wereperformed,wewouldexpectforexamplepeanutandChinesecabbage(Brassicarapa)
miRNAs(bothwithexamplesinmiRBase[15]andPMRD[16])tobedetected.Humanfoods
likechicken,pig,andothermeatsources(manywithmiRNAexamplesavailableonmiRBase)
arealsomissingfromtheresultsofLukasikandZielenkiewicz.Whilenoinformationaboutthe
dietofthehumanpopulationwasprovided[13],pigswereapparentlyfedanundescribedstan-
dardfeed[14]whichprobablydidnotcontainseveraloftheplantslistedinTable1likevine
grape,poplartree,switchgrass,andtobacco.Wewantedtoinvestigatewhetheranymeat
sourceswereavailableinthedataandsearchedforchicken,cow,human,zebrafish,pig,and
dogmiRNAs.Forthisaim,wealignedallavailablematuremiRNAsofthesespeciesfrommiR-
Basetothehumanandporcinereadsallowingnomismatch(Table2).
Weincludedhuman,dogandzebrafishascontrolsandchicken,pig,andcowasexamples
formajorfoodsourcesinChinawherethesamplingwasperformed.TheresultsinTable2
showsignificantnumberofmiRNAsmappingtoallorganismsconsidered.Forpigitcouldbe
arguedthattheevolutionarydistanceisnottoohighandsimilarmiRNAscanberetained,but
thisargumentwouldn’tholdforzebrafishorchicken.Additionally,thepigmiRNAsfoundin
humansamplesshouldsomewhatequalthehumanmiRNAsfoundinpigsamples,iftheywere
evolutionaryconserved.Asthisisnotthecaseandsincepigsamplesaregenerallyoflower
quality(S1Fig),wearguethatthisdifferenceismostlikelyduetocontamination.Insummary,
manyspecies’miRNAswerefoundinbreastmilkalthoughtheyarenotfoodsources(~60%)
and100%ofthecontrolanimalspecies’miRNAswerealsofoundinthebreastmilksamples.
Thisopensthequestionhowtheyweredetectedinbreastmilksampleswithalikelyexplana-
tionbeingcontaminationduringexperimentalprocedure.
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
Table1. PlantmiRNAsreportedtobefoundinhumanand/orporcinesamplesandourassessmentofthembeingfoodsources. Inadditiontoplant
speciesassessedinthestudybyLukasikandZielenkiewicz,weaddedsomeanimals(Table2)indicatedas‘new’.Unlikelyfoodsourcesarehighlightedin
bold.X:cannotbetestedwiththegivendata.‘hsa’and‘ssc’intheNotecolumnmeanthatLukasikandZielenkiewiczreportasignificantidentificationfor
humanand/orpig,respectively.DataalsoavailableasTableAinS2File.
Popularname Species Porcine Human Assessmentofbeingafoodsource Note
samples samples
Coloradoblue Aquilegiacoerulea Found No,allpartsarepoisonous hsa
Columbine
Arabidopsislyrata Arabidopsislyrata Found Leavesarecoatedtopreventeating hsa
Thalecress Arabidopsisthaliana Found Found Notaknownfoodsource,butcloselyrelated hsa
mustardis
Cow Bostaurus Found Found Yes new
Purplefalsebrome Brachypodiumdistachyon Found Similartowheat,butnotamajorfoodsource hsa
Dog Canisfamiliaris Found Found Notinmostpartsoftheworld new
Citrus Citrusparadisi,Poncirus Found Yes hsa
trifoliata
Orange Citrussinensis Found Yes
Citrus Citrustrifoliata Found Found Yes hsa
Zebrafish Daniorerio Found Found No new
Chicken Gallusgallus Found Found Yes new
Soybean Glycinemax Found Found Yes hsa,
ssc
Soybean Glycinesoja Found Found Yes hsa
Human Homosapiens X Found No new
Barley Hordeumvulgare Found Found Yes hsa
Tobacco Nicotianatabacum Found No
Rice Oryzarufipogon Found Yes
Rice Oryzasativa Found Found Yes hsa,
ssc
Switchgrass Panicumvirgatum Found Foodsourceforherbivores,butnotfor ssc
humanorpig
Runnerbean Phaseoluscoccineus Found Yes ssc
Moss Physcomitrellapatens Found No,althoughsomeareedible hsa
Europeansprucetree Piceaabies Found Found Maybeaherbaldrug,butprobablynotinAsia hsa
Poplartree Populuseuphratica Found No,butusedasherbaldrug
Poplartree Populustremuloides Found No,butusedasherbaldrug ssc
Poplartree Populustrichocarpa Found Found No,butusedasherbaldrug hsa,
ssc
Sugarcane Saccharum Found Possiblefoodsourcebutnotexpectedin
detectableamounts
Sorghum Sorghumbicolor Found Found Possiblefoodsourcebutnotexpectedin ssc
detectableamounts
Pig Susscrofa Found X Yes new
Cacaotree Theobromacacao Found Possiblefoodsourcebutnotexpectedin
detectableamounts
Fieldpenny-cress Thlaspiarvense Found Yes,butnotexpectedindetectableamounts ssc
Breadwheat Triticumaestivum Found Yes ssc
Cowpea Vignaunguiculata Found Found Yes hsa,
ssc
Vinegrape Vitisvinifera Found Yes,butwoulditbefedtopigs?
Corn Zeamays Found Found Yes hsa,
ssc
doi:10.1371/journal.pone.0145065.t001
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
Table2. MicroRNAsfoundinhumanandporcinesamplesfromselectedmeatsources. Dog,zebrafishandhumanwereaddedascontrols.Xdenotes
thattheassessmentisnotpossiblewiththegivendata.DataalsoavailableasTableCinS2File.
Popular Species miRBase MicroRNAsinhuman MicroRNAsinporcine Food TotalmiRNAsin
name samples samples Source miRBase
Chicken Gallusgallus gga 160 227 Yes 994
Cow Bostaurus bta 273 394 Yes 793
Pig Susscrofa ssc 229 X Yes 411
Dog Canis cfa 203 262 No 453
familiaris
Human Homo hsa X 1062 No 2588
sapiens
Zebrafish Daniorerio dre 169 209 No 350
doi:10.1371/journal.pone.0145065.t002
RouteforFoodborneMicroRNAstobeDetectableinBreastMilk
Beforeweturntothein-depthanalysisofthehumanandporcinesamples,wemustrecallhow
breastmilkisproducedandconsidertheroutefoodbornemiRNAshavetotaketobefoundin
breastmilk.Thisroutemayencompass:
1. Foodproductionwhichmayinvolvecooking,baking,fermenting,ormanyotherprocessing
stepswhichcouldpotentiallyendangerthestructuralintegrityofsmallRNAs
2. Passagethroughthedigestivetractunharmed.Itisknownthatatleasttheduodenumcon-
tainsnucleasesforRNAandDNA
3. Passagefromthegastrointestinalbarrierintotheblood
4. Uptakebyalveolarcells
5. Secretionfromalveolarcellsasmilk
These5simplifiedstepscontainmanydifferentchemicalenvironmentswithsomeofthem
beingextremelyhostiletosmallRNAs.Therefore,webelievethatifthispathisatallpossible,a
largeamountofmiRNAmustbepresentinthefoodsourceinordertocreateameasurable
titerinbreastmilk.ThemiRNAcounts,asprovidedbyLukasikandZielenkiewicz,whichwere
measuredfrom20–30mlbreastmilk,were,inouropinion,toolow(24clusteredreadsforthe
highestabundantmiRNA)tobeabletosignificantlyaffectaninfantinanyform.Webelieve
thatthemiRNAtiterinafoodsourcemustbesignificantlyhigherthanthatbecausemany
miRNAswillbedegradedalongthedigestiveprocess.Forthetransferintobreastmilk,itis
importanttonote,thatanyhumancellcanuptakethecirculatingmiRNAsfromtheblood
stream,significantlyreducingtheconcentrationinthealveolarcells.Finally,thecompletecon-
tentsofthesecellsarenotexcretedasmilkleadingtoanadditionaldecreaseoftheamountof
smallRNAsdetectabletherein.
Itshould,however,benotedthatinfantsdonothaveawell-developedgastrointestinalbar-
rierthuspotentiallyallowinglargermoleculestopassdirectlyintotheblood(forexamplethe
mother’santibodies).ThiswouldopenopportunitiesformiRNAstomoreeasilypassintothe
bloodstreamifthereareanyinthemilk;perhapsspecies-specificmiRNAs.Weconcludethat
infantsmightbeabletomoreeasilypick-upmiRNAsfromfood,butwouldnotbeaffectedby
thesmallamountofroughly1miRNApermlbreastmilkasreported.ForthetransferofmiR-
NAsfromfoodintobreastmilk,weconcludethatitmayonlybepossibleiftheyareavailable
inexcessiveamounts,anotionsupportedbythestudyofRWLusk[11].Inthefollowingwe
willelaborateonwhythesemiRNAsarefoundinhumanandporcinebreastmilksamples.
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
EvidenceforMessengerRNAsinHumanandPorcineSamples
ItcanbehypothesizedthatfoodbornemiRNAscouldbeenrichedinbreastmilksincetheyare
rathersmall.Wewondered,however,whetherthereareonlymiRNAsinthebreastmilksamples
orwhetherotherRNAspeciescouldalsobefound.Therefore,wemappedreadstoavailabletran-
scriptsfromN.tabacum(notafoodsource),A.thaliana(unlikelyafoodsource),andO.sativa(a
staplefood).First,sequencingadapterswereremovedfromthereads.Thenthereadswerequality
trimmedandalignedtotheirrespectivegenome(human/pig).Forthehumansamplesonaver-
agelessthan6%ofreadsremainedunaligned,butfortheporcinesamplesonaverageabout57%
ofthereadsdidnotaligntopigprobablymostlyduetolowqualityofsequencingresults(com-
paresequencingqualityresultsinS1FigandTableFinS2File).Weretrievedabout3000tran-
scriptsfortobaccofromNCBI,~7000forA.thalianafromTAIR,andmorethan13000forrice
fromPlantGDB(TablesJ,N,andRinS3,S4andS5Files).Inthecombinedhumansamples
about200(~7%)andinthecombinedporcinesamples6880(~98%)A.thalianatranscriptswere
detectedwhileabout80weredetectedinboth(TablesK,O,andSinS3,S4andS5Files).Similar
numbersformappabletranscriptswerefoundfortobacco(~18%;~90%)andrice(~9%;~94%).
Onaverageabout0.7,1.2,and1.8%ofthereadsalignedtoA.thaliana,tobaccoandrice,respec-
tively.Theaboveassessmentonlyshowedthatreadsmappedtotranscripts,butitisknownthat
somemiRNAscanoriginatefromcodingsequencesandthusthesemappedreadscouldrepresent
miRNAs.Therefore,weassessedthesequencecoveragethatcanbeachievedforthedetectedtran-
scripts(TablesJ,NandRinS3,S4andS5Files).Here,thesequencecoverageistheratioofnucle-
otidesforwhichatleastonereadalignedtothetranscriptdividedbythenumberofnucleotides
inthetranscript.Table3showsanexcerptforA.thalianafromthecompletedata(TableJinS3
File)anditisseenthatsometranscriptshavesequencecoverageofcloseto90%.Onaveragefor
allsamples2,2,and4transcriptswithcoverageofover80%werefoundfortobacco,A.thaliana,
andrice,respectively(TablesL,P,andTinS3,S4andS5Files).Thedistributionforsequence
coverageamongsamplescanbeseeninFig1andsimilarfiguresfortheotherspeciesareavailable
inS1File(FigsA–CinS1File).Averagedoverallsamples,about1%(O.sativa),~2%(N.taba-
cum),and~2%(A.thaliana)ofreadshaveahigherthan30%sequencecoverage(TablesL,P,
andTinS3,S4andS5Files).Onaverage,wefound276tobaccotranscriptsinhuman(682inpor-
cine),74A.thalianatranscriptsinhuman(1528porcine),and347ricetranscriptsinhuman(in
porcine3326)samples.Twohumanbreastmilksamples(SRR346518,SRR346519)andonepig
sample(SRR445993)containmuchlessmatchestotranscriptsforallthreespecies(0.1–0.3fold
ofremainingsamplesTablesK,O,andSinS3,S4andS5Files).
Onlymappedtranscriptswereusedtocalculatethedistribution(Fig1;TableNinS4File)
anditseemsthattranscriptshadlittlecoverageonaverage.Thiscouldbeattributedtoanover-
alllownumberofreadsofthecontamination.Interestingly,someshowedveryhighcoverage
similartotheresultsforA.thaliana(Table3).SimilarfiguresforO.sativa(FigCinS1File)
andA.thaliana(FigAinS1File)areavailableandtheunderlyingdataispresentedinTablesR
andJinS5andS3Files,respectively.
Highsequencecoverageofupto94%forforeigntranscriptsarenotexpectedtobefoundin
breastmilkexceptforfromthemicrobiomeandspeciesspecificones.Forallsamplesseveral
transcriptsshowedhigherthan80%sequencecoverage,whichisenormousconsideringthat
onlyfewreadsareactuallycontaminatingtherespectivesamples.Threesamplesshowless
alignmentandlowersequencecoverageoverall,butemailinquiriestodeterminewhyremained
unanswered.Fromtheseresults,weconcludethatmRNAsmusthavecontaminatedthesam-
ples,whichdoesnotexcludethatmiRNAs,whichmayoriginatefromanypartofthegenome
[17],arealsoamongthecontaminantsorstemfromsomeofthemRNAs.However,itseemed
clearfromtheseresultsthatmiRNAswerenotenrichedinbreastmilk.
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
8
h 9
ng 9
foundiVeryhi RR445 0.51 0.33 0.34 0.19 0.07 0.22 0.17 0.16 0.25 0.16 0.13
bey. S
datacanespectivel R445997 0.26 0.19 0.20 0.12 0.07 0.17 0.10 0.11 0.04 0.04 0.09
er R
ets, S
ThecomplandS5File R445996 0.14 0.20 0.21 0.09 0.03 0.23 0.11 0.08 0.15 0.07 0.13
g).S4 SR
mnsforpiNandRin R445995 0.18 0.12 0.13 0.03 0.20 0.07 0.04 0.05 0.03 0.03
u R
oles S
andlast8cdatainTablIinS2File. RR445994 0.24 0.16 0.18 0.07 0.03 0.23 0.06 0.05 0.30 0.05 0.03
fourcolumns;dtheircompleteailableasTable SRR445993S 0.36 0.29 0.28 0.18 0.14 0.38 0.09 0.14 0.09 0.07 0.10
Arabidopsisthalianafoundinhuman(firstandricecanbefoundinTableIinS2FileanNtabacumOsativamationfor.and.isalsoav 8SRR346519SRR445991SRR445992 0.080.440.37 0.150.320.32 0.210.300.33 0.090.140.14 0.070.200.05 0.240.380.46 0.090.140.13 0.180.170.14 0.080.210.25 0.060.090.09 0.110.100.06
mcoor 51
sfroobacarinf R346 0.24 0.17 0.20 0.11 0.18 0.33 0.07 0.14 0.07 0.07 0.03
criptafortsimil SR
ansdatand 517
oftrmilardata R346 0.82 0.72 0.59 0.55 0.59 0.54 0.61 0.60 0.57 0.40 0.38
coverageoneandsiThesame 6516SR 9 3 5 3 1 0 7 5 7 0 8 45065.t003
Table3.PercentreadTableJinS3File.Thiscoveragesarebolded. TranscriptSRR34 gi|424697940.8 gi|1107401280.6 gi|158101990.5 gi|202593980.5 gi|202593590.5 gi|1107406430.5 gi|204660660.4 gi|424682090.4 gi|424677810.3 gi|1162943700.3 gi|424679490.2 doi:10.1371/journal.pone.01
PLOSONE|DOI:10.1371/journal.pone.0145065 January29,2016 7/17
Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
Fig1.ReadCoverageAcrossTranscripts.DistributionoftranscriptcoverageforNicotianatabacumin
human(first4)andporcine(last8)samples.DatacanbefoundinTableNinS4File.
doi:10.1371/journal.pone.0145065.g001
SomeTranscriptsareSharedAmongSamples
AfteridentifyingreadsmappingtomRNAsinthebreastmilksamples,wewereinterestedin
whetherthesamemRNAswouldbesharedamonghuman(4),porcine(8),orall(12)samples.
ForA.thalianaabout200transcriptsweredetectedinhumansamples,andoutofthose21
(~10%)weresharedamongallhumansamples(TableKinS3File).Assumingrandomdraw-
ing,thechanceforfindingaparticulartranscriptsharedamongsamplesis:
n n n n
p ¼ 1(cid:2) 2(cid:2) 3(cid:2) X
shared N N N N
wheren isthenumberoftranscriptsfoundforthesamplexandNisthenumberofavailable
x
transcripts.ForA.thalianatheprobabilitythathuman,pig,orallsamplesshareaparticular
transcriptis7(cid:3)10−9,1(cid:3)10−6,and8(cid:3)10−15,respectively.Forricethisisquitesimilarwith
2(cid:3)10−8,3(cid:3)10−6,and7(cid:3)10−14,butfortobaccotheprobabilityisslightlyhigherwith3(cid:3)10−5,
1(cid:3)10−6,and5(cid:3)10−11sincetherearelessavailabletranscripts.Despitethelowprobabilities,A.
thalianaandtobaccohave10transcriptsthataresharedamongall12sampleswhilericehas4
(TablesK,OandSinS3,S4andS5Files).ThiscalculationwasonlyforonemRNA,butsets
ofmRNAscanbesharedamongsamples,aswell,whichcanbeanalyzedusingPearsoncorrela-
tion(Fig2).AhighercorrelationmeansmoremRNAsaresharedbetweenthesamples.Fig2
providesanexamplecorrelationforriceandsimilarfiguresforA.thalianaandtobaccoare
availableinS1File(FigsD–FinS1File).Itisinterestingtonotethathighcorrelationsarenot
onlyfoundbetweensamplesfromthesamespecies,butalsobetweenorganisms.Forexample,
thenumberofmappedA.thalianatranscriptssharedbetweensampleSSR346517(human)
andSRR445993(pig)is27.IntheMaterialsandMethodsSectionweshowhowtocalculatethe
probabilityforsharedtranscriptsamongsamplesandconcludethatitisextremelylow
(5(cid:3)10−15).TablesM,Q,andUinS3,S4andS5Filesshowthenumberofsharedtranscripts
betweensamplesandprovidetheprobabilityforthattobebychance.
FindingmRNAsinthebreastmilksamplesisproblematicforthehypothesisthatfoodborne
xeno-miRNAs,performingcross-kingdomregulations,aredeliberatelyenrichedtherein.Addi-
tionally,weanalyzedwhetherthemRNAsthatwereidentifiedinthedifferentsamplesare
sharedamongthem.Despitethelowprobabilityforthattobepossible,wefoundhighcorrela-
tionsbetweensamples(intraandinter-species).Also,ifthemRNAswerefoodborne,wewould
expectsignificantdifferencesatleastamongthehumansamples.Finally,wewouldexpectno
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
Fig2.CorrelationofTranscriptsBetweenSamples.CorrelationamongtranscriptsofOryzasativafoundin
humanandporcinesamples.Thefirst4samplesacrossthetopandthefirst4rowsfromtopcorrespondto
humansamples;theremainderisofpigorigin.Highcorrelationisvisualizedviadarkercolorandlarger
circles.CorrespondingfiguresforA.thaliana(FigDinS1File)andN.tabacum(FigEinS1File).
doi:10.1371/journal.pone.0145065.g002
significantcorrelationbetweenhumanandporcinesamples,butformanyofthesampleswe
foundhighcorrelations(Fig2,FigsBandCinS1File).Apracticalexplanationforthisisthat
theRNAswerecontaminationswhichwereintroducedaftersampling.
AllIdentifiedOrganismswereHandledintheSameInstitute
TheprevioussectionshavedetailedhowweidentifiedRNAsinthesamplesanddetermined
thattheywerepotentiallycontaminants.We,therefore,wonderedwhethertherewasanyevi-
denceelucidatinghowtheseplantRNAscancontaminatethehumanandporcinebreastmilk
samples.AllsamplesweremeasuredattheSichuanAgriculturalUniversity,Sichuan,China
andweinquiredviaemailwhethersuchcontaminationswerepossible,butfailedtoreceivean
answer.SoweturnedtoThomsonReuters’WebofKnowledge(WOK)toinvestigatewhether
anystudyinrespecttothepossiblecontaminatingorganismswaspublishedduringthetime
periodof2010–2013.Ifpublishedduringthisperiod,thesamplesforthosepublicationscould
havebeenprocessedinthesamelabatthesametimeasthehumanandporcinesamples.We
foundsomeevidenceandmanyoftheorganismsreportedtocontributeRNAstothesamples
werecovered,butnotall.ThereforeweturnedtoGoogleScholar,whichcoversmorejournals
thantheWOKandfoundpublicationsforallspeciesduringtheselectedtimeperiod(Table4).
Table4summarizestheplantspeciespreviouslystudiedandtheanimalspecieswespecifi-
callyaddedtotheanalysis.Weobservedacompoundcorrelationamongthenumberofthe
studiesfoundforaspecies,thenumberofmiRNAsknownforaspecies,andtheamountof
miRNAsfoundinhumanandporcinesamplesforthatspecies(Table5andTableDinS2File).
Therowshighlightedingray(Table5),wereaddedbyustotheanalysisandwedidnotper-
formclusteringofreads,whichisthereasonwhytheircountscannotbecomparedtothe
countsfortheplantsbelowandwhytheyaregenerallylarger.Also,animalmiRNAswere
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Xeno-MicroRNAsReportedinBreastMilkAreArtefacts
Table4. HitsonGoogleScholarwhensearchingforthenameoftheinstitutethatperformedNGSlimitedtothetimeperiodof2010–2013and
requiringanadditionalmatchtothespeciesthatwerereportedtocontributemiRNAstothesamples. Wereasonthatastudypublishedinthistime
periodcouldhavebeenhandledatthesametimeasthehumanandporcinebreastmilksamples.ThenumberofGoogleScholarhitsthatcontainsequencing
orNGSintitleorabstractisindicatedinparentheses.Pleasenote,however,thatcontaminantscanbeintroducedduringanystepoftheexperimentalproce-
dureandnotjustduringsequencingand,therefore,thehitsonGoogleScholararethemostindicativemeasureforthisanalysis.Dataisalsoavailableas
TableBinS2File.
PopularPlant PlantSpecies Porcine Human Hitson Queryresultslimitedtothetimeperiod2010–2013
Name Samples Samples Google
Scholar
Rice Oryzasativa Found Found 1430(164) "sichuanagriculturaluniversity""Oryzasativa"
Corn Zeamays Found Found 1170(115) "sichuanagriculturaluniversity""zeamays"
Breadwheat Triticumaestivum Found 927(133) "sichuanagriculturaluniversity""Triticumaestivum"
Thalecress Arabidopsisthaliana Found Found 607(179) "sichuanagriculturaluniversity""Arabidopsisthaliana"
Soybean Glycinemax Found Found 482(42) "sichuanagriculturaluniversity""glycinemax"
Barley Hordeumvulgare Found Found 316(61) "sichuanagriculturaluniversity""Hordeumvulgare"
Tobacco Nicotianatabacum Found 207(35) "sichuanagriculturaluniversity""Nicotianatabacum"
Sorghum Sorghumbicolor Found Found 204(29) "sichuanagriculturaluniversity""Sorghumbicolor"
Poplartree Populuseuphratica Found 193(11) "sichuanagriculturaluniversity""Populuseuphratica"
Vinegrape Vitisvinifera Found 173(35) "sichuanagriculturaluniversity""Vitisvinifera"
Sugarcane Saccharum Found 167(14) "sichuanagriculturaluniversity""Saccharum"
Rice Oryzarufipogon Found 132(8) "sichuanagriculturaluniversity""Oryzarufipogon"
Citrus Citrussinensis Found 122(10) "sichuanagriculturaluniversity""Citrussinensis"
Soybean Glycinesoja Found Found 113(7) "sichuanagriculturaluniversity""Glycinesoja"
Pig Susscrofa X Found 106(59) "sichuanagriculturaluniversity""Susscrofa"
Human Homosapiens Found X 85(49) "sichuanagriculturaluniversity""Homosapiens"
Chicken Gallusgallus Found Found 84(42) "sichuanagriculturaluniversity""Gallusgallus"
Cow Bostaurus Found Found 81(44) "sichuanagriculturaluniversity""Bostaurus"
Switchgrass Panicumvirgatum Found 68(5) "sichuanagriculturaluniversity""Panicumvirgatum"
Zebrafish Daniorerio Found Found 55(26) "sichuanagriculturaluniversity""Daniorerio"
Poplartree Populustrichocarpa Found Found 46(19) "sichuanagriculturaluniversity""Populustrichocarpa"
Citrus Citrusxparadisix Found 40(7) "sichuanagriculturaluniversity"("Citrusxparadisi"OR
Poncirustrifoliata "Poncirustrifoliata")
Purplefalse Brachypodium Found 33(18) "sichuanagriculturaluniversity""Brachypodium
brome distachyon distachyon"
Cowpea Vignaunguiculata Found Found 32(2) "sichuanagriculturaluniversity""Vignaunguiculata"
Europeanspruce Piceaabies Found Found 24(4) "sichuanagriculturaluniversity""Piceaabies"
tree
Moss Physcomitrellapatens Found 19(9) "sichuanagriculturaluniversity""Physcomitrellapatens"
Dog Canisfamiliaris Found Found 15(13) "sichuanagriculturaluniversity""Canisfamiliaris"
Runnerbean Phaseoluscoccineus Found 13(0) "sichuanagriculturaluniversity""Phaseoluscoccineus"
Poplartree Populustremuloides Found 11(1) "sichuanagriculturaluniversity""Populustremuloides"
Fieldpenny-cress Thlaspiarvense Found 10(0) "sichuanagriculturaluniversity""Thlaspiarvense"
Cacaotree Theobromacacao Found 9(6) "sichuanagriculturaluniversity""Theobromacacao"
Arabidopsislyrata Arabidopsislyrata Found 4(4) "sichuanagriculturaluniversity""Arabidopsislyrata"
ColoradoBlue Aquilegiacoerulea Found 1(0) "sichuanagriculturaluniversity""Aquilegiacoerulea"
Columbine
Orange Citrustrifoliata Found Found 1(1) "sichuanagriculturaluniversity""Citrustrifoliata"
doi:10.1371/journal.pone.0145065.t004
establishedusingdatafrommiRBasewhereasplantmiRNAswereestablishedusingdatafrom
PMRD[16].Specieswithmanypublishedstudies(highlikelihoodofconcurrenthandling)and
manyknownmiRNAs(highlikelihoodforspuriousassignment)tendtobehigherinthelist
andhavemorehitsintheinvestigatedsamples.Thereareoutlierswhichmaypossiblybedue
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