advantages and limitations of qPcR

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MINIREVIEWAdvantagesandlimitationsofquantitativePCR(Q-PCR)-basedapproachesinmicrobialecologyCindyJ.Smith&A.MarkOsbornDepartmentofAnimalandPlantSciences,UniversityofSheffield,WesternBank,Sheffield,UKCorrespondence:A.MarkOsborn,AbstractDepartmentofAnimalandPlantSciences,QuantitativePCR(Q-PCRorreal-timePCR)approachesarenowwidelyappliedUniversityofSheffield,WesternBank,SheffieldS102TN,UK.Tel.:144114222inmicrobialecologytoquantifytheabundanceandexpressionoftaxonomicand4626;fax:1441142220002;e-mail:functionalgenemarkerswithintheenvironment.Q-PCR-basedanalysescombinea.m.osborn@sheffield.ac.uktraditionalend-pointdetectionPCRwithfluorescentdetectiontechnologiestorecordtheaccumulationofampliconsinrealtimeduringeachcycleofthePCRReceived20June2008;revised10Octoberamplification.Bydetectionofampliconsduringtheearlyexponentialphaseofthe2008;accepted24October2008.PCR,thisenablesthequantificationofgene(ortranscript)numberswhentheseFirstpublishedonlineDecember2008.areproportionaltothestartingtemplateconcentration.WhenQ-PCRiscoupledwithaprecedingreversetranscriptionreaction,itcanbeusedtoquantifygeneDOI:10.1111/j.1574-6941.2008.00629.xexpression(RT-Q-PCR).ThisreviewfirstlyaddressesthetheoreticalandpracticalimplementationofQ-PCRandRT-Q-PCRprotocolsinmicrobialecology,high-Editor:MichaelWagnerlightingkeyexperimentalconsiderations.Secondly,wereviewtheapplicationsofKeywords(RT)-Q-PCRanalysesinenvironmentalmicrobiologyandevaluatethecontribu-Q-PCR;RT-Q-PCR;16SrRNAgene;mRNA.tionandadvancesgainedfromsuchapproaches.Finally,weconcludebyofferingfutureperspectivesontheapplicationof(RT)-Q-PCRinfurtheringunderstandinginmicrobialecology,inparticular,whencoupledwithothermolecularapproachesandmoretraditionalinvestigationsofenvironmentalsystems.aspyrosequencing(Marguliesetal.,2005;Edwardsetal.,Introduction2006)nowdwarfPCR-basedsequencestudiesintermsofTheapplicationofPCRincombinationwiththeextractionsequencecoverage,theabilityofthePCRtospecificallyofnucleicacids(DNAandRNA)fromenvironmentaltargetparticulartaxonomicorfunctionalmarkersfrommatriceshasbeencentraltothedevelopmentofculture-domaindowntostrainorphylotypelevelsmeansthatindependentapproachesinmicrobialecology.Thesemeth-PCRwillremainaninvaluablemethodinthemolecularods,whichhavebeenappliedsincetheearly1990s(e.g.microbialecologiststoolbox.Nevertheless,PCRhasinher-Giovannonietal.,1990),enablingtheanalysisofthetotalentlimitations(vonWintzingerodeetal.,1997),particularlymicrobialcommunitiespresentwithinenvironmentalsys-thosethatresultinbiasesinthetemplatetoproductratiosoftems,haverevolutionizedourunderstandingofmicrobialtargetsequencesamplifiedduringPCRfromenvironmentalcommunitystructureanddiversitywithintheenvironment.DNA(Suzuki&Giovannoni,1996;Polz&Cavanaugh,Couplingenvironmentalnucleicacidisolationtosubse-1998),withsuchamplificationbiasesfoundtoincreasewithquentPCRamplificationofbothtaxonomic(i.e.rRNA)increasingnumbersofPCRcycles.Theselimitationspre-andfunctionalgenemarkersandincombinationwithDNAsentedasignificantchallengetomicrobialecologistswhofingerprinting-andsequencing-basedanalyseshasenabledwereinterestedindeterminingtheabundanceofindividualdescriptionofthehithertouncharacterizedmajorityofgenespresentinenvironmentalsamples.Tocircumventsuchenvironmentalmicroorganisms(Headetal.,1998)drivingchallenges,anadaptationofthePCRmethoddevelopedbythediscoveryofnewmicrobiallineagesandenablingtheHollandetal.(1991)utilizingtheso-called50nucleasedescriptionofgeneticdiversityinawealthoffunctionalassaywasappliedtoquantifytarget16SrRNAgenesgenemarkers(Larkinetal.,2005).Althoughrecentlydevel-amplifiedfromenvironmentalDNAbyPCR(Beckeretal.,opedultra-high-throughputsequencingtechnologiessuch2000;Suzukietal.,2000;Takai&Horikoshi,2000).Thisc2008FederationofEuropeanMicrobiologicalSocietiesFEMSMicrobiolEcol67(2009)6–20PublishedbyBlackwellPublishingLtd.Allrightsreserved ApplicationofQ-PCRinmicrobialecology7developmenthadbeenfacilitatedbytheearliercombinationproductviaacorrespondingincreaseinthefluorescentofthe50nucleaseassaydevelopedbyHollandetal.(1991)signalassociatedwithproductformationduringeachcyclewithfluorescencedetectionfollowingcleavageofaninternalinthePCR.Quantificationofgene(ortranscript)numbersTM(TaqMan)DNAprobe(Livaketal.,1995),enablingtheisdeterminedduringtheexponentialphaseofthePCRaccumulationofampliconstobemonitoredaftereachcycleamplificationwhenthenumbersofampliconsdetectedare(inreal-time)andhencefacilitatingquantitativedetermina-directlyproportionaltotheinitialnumbersoftargettionoftheinitialtemplategene(ortranscript)numbers.sequencespresentwithintheenvironment(discussedinQuantitative-PCRorQ-PCR(oftenreferredtoasreal-moredetailinTargetquantification).QuantificationofthetimePCR)isnowwidelyusedinmicrobialecologytotargetgeneduringexponentialamplificationavoidspro-determinegeneand/ortranscriptnumberspresentwithinblemsthatareassociatedwithso-calledend-pointPCRenvironmentalsamples.ThetargetspecificityofanyQ-PCR(inwhichampliconsareonlyanalysedaftercompletionofassayisdeterminedbythedesignoftheprimers(andinthefinalPCRcycle).Inend-pointPCR,theproportionssomecasesaninternalprobe),allowingquantificationofofnumericallydominantampliconsdonotnecessarilytaxonomicorfunctionalgenemarkerspresentwithinareflecttheactualabundancesofsequencespresentwithinmixedcommunityfromthedomainleveldowntothetheenvironmentduetotheinherentbiasesofPCRthatquantificationofindividualspeciesorphylotypes.Q-PCRareassociatedwithamplificationoftargetsfrommixedhasbeenshowntobearobust,highlyreproducibleandtemplatecommunityDNA(Reysenbachetal.,1992;Suzukisensitivemethodtoquantitativelytrackphylogeneticand&Giovannoni,1996;Polz&Cavanaugh,1998).Moreover,functionalgenechangesacrosstemporalandspatialscalesQ-PCRthatusesfluorescence-baseddetectionoffersgreaterundervaryingenvironmentalorexperimentalconditions.sensitivityandenablesdiscriminationofgenenumbersMoreover,thequantitativedatageneratedcanbeusedtoacrossawiderdynamicrangethanisfoundwithend-pointrelatevariationingeneabundancesand/orlevelsofgenePCR;forexampletwofoldchangesintargetconcentrationexpression(intermsoftranscriptnumbers)incomparisoncanbediscriminatedusingQ-PCR.Beforethedevelopmentwithvariationinabioticorbioticfactorsand/orbiologicaloffluorescence-basedQ-PCR-basedmethods,twoalterna-activitiesandprocessrates.TheprovisionofQ-PCRdatativePCR-basedmethodsforgenenumberquantificationsetsthatdescribetheabundanceofspecificbacteriaorgeneshadbeendeveloped,namelycompetitivePCR(Diviaccotocomplementotherquantitativeenvironmentaldatasetsetal.,1992)andlimitingdilutionsormostprobablenumberisofincreasingimportanceinmicrobialecologyasit(MPN)-PCR(Skyesetal.,1992).However,thesemethodsfurthersunderstandingoftherolesandcontributionsofaretime-andresource-consuming,requiringpost-PCRparticularmicrobialandfunctionalgroupswithinecosys-analysis,andhavenowlargelybeenreplacedbyfluores-temfunctioning.Furthermore,reversetranscription(RT)cence-basedQ-PCRmethods.analysesarenowincreasinglycombinedwithQ-PCRmethodsinRT-Q-PCRassays,offeringapowerfultoolforFluorescencedetectionchemistriesusedquantifyinggeneexpression(intermsofnumbersofrRNAtodetecttemplateamplificationduringandmRNAtranscripts)andrelatingbiologicalactivitytoQ-PCRecologicalfunction.Inthisreview,wefirstlydiscussthemechanisticaspectsofQuantitativereal-timePCRworksinessentiallythesameQ-PCRandRT-Q-PCRmethodologies,hereafterdefinedmannerasend-pointPCR,i.e.multipleamplificationcyclescollectivelyas(RT)-Q-PCR,andhighlightthekeyexperi-inwhichtemplateDNAisinitiallydenatured,followedbymentalconsiderationsinthedesignandimplementationofannealingofoligonucleotideprimerstargetingspecific(RT)-Q-PCRprotocolsandtheanalysisofresultantdatasequences,followedbysubsequentextensionofacomple-sets.Secondly,weexploretheapplicationof(RT)-Q-PCRmentarystrandfromeachannealedprimerbyathermo-approachesinmicrobialecology,andfinallywediscusshowstableDNApolymerase,resultinginanexponentialincreasethesemethodscanbeappliedtogetherwithothermolecularinampliconnumbersduringthePCR.However,incontrastandalsoconventionalapproachestoprovideanincreasedtoend-pointPCR,theincreaseinampliconnumbersisunderstandingofmicroorganismswithinenvironmentalrecordedinreal-timeduringthePCRviadetectionofasystems.fluorescentreporterthatindicatesampliconaccumulationduringeverycycle.Tworeportersystemsarecommonlyused,namely,theintercalatingSYBRgreenassay(WittwerAdvantagesofQ-PCRovertraditionalend-etal.,1997)andtheTaqManprobesystem(Hollandetal.,pointPCR1991;Livaketal.,1995).Q-PCRapproachescombinethedetectionoftargettemplateSYBRgreenbindstoalldouble-strandedDNAviainter-withquantificationbyrecordingtheamplificationofaPCRcalationbetweenadjacentbasepairs.WhenboundtoDNA,FEMSMicrobiolEcol67(2009)6–20c2008FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.Allrightsreserved 8C.J.Smith&A.M.Osbornafluorescentsignalisemittedfollowinglightexcitation(Fig.1995).Thecloseproximityontheprobeofthequencher1a).AsampliconnumbersaccumulateaftereachPCRcycle,moleculetothefluorophorepreventsitfromfluorescingthereisacorrespondingincreaseinfluorescence.Becauseduetofluorescentresonanceenergytransfer.DuringtheSYBRgreenbindstoalldouble-strandedDNA,itisessentialannealingstepofeachcycleofthePCR,primersandthetouseprimerpairsthatarehighlyspecifictotheirtargetintactprobebindtotheirtargetsequences.Duringsubse-sequencetoavoidgenerationofnonspecificproductsthatquenttemplateextension,the50exonucleaseactivityofthewouldcontributetothefluorescentsignal,resultinginanTaqpolymeraseenzymecleavesthefluorophorefromoverestimationofthetarget.ExtensiveoptimizationoftheTaqManprobeandafluorescentsignalisdetectedasprimerconcentrationsusedinSYBRgreenQ-PCRassaysthefluorophoreisnolongerincloseproximitytothemayberequiredtoensurethatonlythetargetedproductisquencher(Fig.1b).Amplificationofthetemplateisthusformed.Primerpairsthatexhibitself-complementaritymeasuredbythereleaseandaccumulationofthefluoro-shouldalsobeavoidedtopreventprimerdimerformation.phoreduringtheextensionstageofeachPCRcycle.TheApost-PCRdissociation(melting)curveanalysisshouldbeadditionalspecificityaffordedbythepresenceofthecarriedouttoconfirmthatthefluorescencesignalisTaqManprobeensuresthatthefluorescentsignalgeneratedgeneratedonlyfromtargettemplatesandnotfromtheduringQ-PCRisderivedonlyfromamplificationoftheformationofnonspecificPCRproducts.Duringadissocia-targetsequence.MultipleTaqManprobesandprimersetstioncurve,thedouble-strandedtemplateisheatedoveracanbeusedindifferentQ-PCRassaystodifferentiatetemperaturegradientandfluorescencelevelsaremeasuredbetweencloselyrelatedsequences(Smithetal.,2007),orateachdiscretetemperaturepoint.Asthedouble-strandedalternatively,probescanbelabelledwithdifferentfluoro-templateisheated,itdenatures,resultinginacorrespondingphores,facilitatingthedevelopmentofmultiplexQ-PCRdeclineinfluorescenceduetoSYBRgreendissociationfromprotocolswherebydifferenttargetscanbecoamplifiedandthedouble-strandedproduct(Giglioetal.,2003;Gonzalez-quantifiedwithinasinglereaction(Neretinetal.,2003;Escalonaetal.,2006).Thetemperatureatwhich50%oftheBaldwinetal.,2003,2008).Forexample,Baldwinetal.double-strandedtemplateisdenaturedcanbeusedto(2003)developedamultiplexQ-PCRassaytargetingaconfirmthatthetemplatebeingtargetedispresent,alongnumberofdifferentaromaticoxygenasegenesusingbacter-withthepresenceofothernonspecifictemplateandprimerialstrainsandthensubsequentlyappliedtheassaytodimersinmuchthesamewayasagarosegelelectrophoresissimultaneouslyquantifyaromaticoxygenasegenesincon-ofanend-pointPCRproductisused.taminatedgroundwater(Baldwinetal.,2008).TaqManTheTaqManprobemethodutilizesafluorescentlyprobesare,however,amoreexpensiveoptionthanusinglabelledprobethathybridizestoanadditionalconservedSYBRgreenchemistryandtheformerrequiresthepresenceregionthatlieswithinthetargetampliconsequence.TheofanadditionalconservedsitewithintheshortampliconTaqManprobeisfluorescentlylabelledatthe50endandsequencetobepresent.Identificationofthreeconservedcontainsaquenchermoleculeatthe30end(Livaketal.,regionswithinashortregion(typicallyc.o100bp)maynotFig.1.Real-timePCRchemistries:(a)SYBRgreendetection.SYBRgreenbindstoalldouble-strandedDNAandemitsafluorescentsignal.Initsunboundstate,SYBRgreendoesnotfluoresce.Templateamplificationisthereforemeasuredineachcyclebythecorrespondingincreaseinfluorescence.(b)TaqMan(50nuclease)sassayusingTaqManprobes.Duringannealing,theTaqManprobeandprimersbindtothetemplate.WhentheTaqManprobeisintact,energyistransferredbetweenthequencherandthereporter;asaresult,nofluorescentsignalisdetected.AsthenewstrandissynthesizedbyTaqpolymerase,the50exonucleaseactivityoftheenzymecleavesthelabelled50nucleotideoftheprobe,releasingthereporterfromtheprobe.Onceitisnolongerincloseproximity,thefluorescentsignalfromtheprobeisdetectedandtemplateamplificationisrecordedbythecorrespondingincreaseinfluorescence.c2008FederationofEuropeanMicrobiologicalSocietiesFEMSMicrobiolEcol67(2009)6–20PublishedbyBlackwellPublishingLtd.Allrightsreserved ApplicationofQ-PCRinmicrobialecology9alwaysbepossible,especiallywhenprimer/probecombina-ciallyavailableinstrumentstocarryoutQ-PCR,eachwithtionsarebeingdesignedtotargetdivergentgenesequences.itsownassociatedsoftware.Currently,thereisconsiderableMorerecentadvancesinTaqManprobetechnologyhavedebateastowhichalgorithmsarethebestusedtoanalyseinvolvedtheintroductionoftheminorgroovebinderQ-PCRdata(reviewedinRebrikov&Trofimov,2006).All(MGB)probe(Kutyavinetal.,2000).TheMGBmoleculeistheQ-PCRplatformscollectfluorescentdatafromeveryattachedtothe30endoftheprobeandessentiallyfoldsbackamplificationcycleandtheincreaseinfluorescenceisontotheprobe.Thisnotonlyincreasesthestabilityoftheplottedagainstthecyclenumber,resultinginthetypicalprobe,butallowsthedesignofshorterprobes(1320bp)amplificationcurveshowninFig.2.TheQ-PCRamplifica-thanarerequiredfortraditionalTaqManprobes(2040bp),tioncurvecanbesubdividedintofourstages,namelywhileatthesametime,maintainingtherequiredhybridiza-backgroundnoise,wherethebackgroundfluorescencestilltionannealingtemperature.exceedsthatderivedfrominitialexponentialtemplateaccumulation,exponentialamplification,linearamplifica-tionandaplateaustage.DuringtheexponentialphaseoftheTargetquantificationusingthecycleamplification,theamountoftargetamplifiedispropor-threshold(Ct)methodtionaltothestartingtemplateanditisduringthesecyclesIrrespectiveofthefluorescencechemistryused,quantifica-thatgenenumbersarequantifiedusingtheCtmethod.ThetionofthetargettemplateDNAiscarriedoutinessentiallyCtisreachedwhentheaccumulationoffluorescence(tem-thesamemanner.Thereareanumberofdifferentcommer-plate)issignificantlygreaterthanthebackgroundlevelFig.2.Q-PCRamplificationfromknownconcentrationsoftemplateDNAtoconstructstandardcurvesforquantificationofunknownenvironmental481samples.(a)Logplotoftheincreaseinfluorescencevs.cyclenumberofDNAstandardsrangingfrom110to11016SrRNAgeneampliconsmL.(b)LinearplotindicatingthethreephasesofaPCRamplification,thecorrespondingCtvaluesforeachoftheamplifiedstandardsandfortheNTC.(c)SimplelinearregressionoftheCtvalues(fromb)vs.logoftheinitialrRNAgenenumber.Q-PCRdescriptorsareshown(boxed).FEMSMicrobiolEcol67(2009)6–20c2008FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.Allrightsreserved 10C.J.Smith&A.M.Osborn(Heidetal.,1996).Duringtheinitialcycles,thefluorescencecurvegeneratedfromamplificationofthetargetgenesignalduetobackgroundnoiseisgreaterthanthatderivedpresentatarangeofinitialtemplateconcentrations,andfromtheamplificationofthetargettemplate.OncetheCtthentheCtvaluesforeachtemplateconcentrationarevalueisexceeded,theexponentialaccumulationofproductdetermined.Subsequently,asimplelinearregressionofcanbemeasured.WhentheinitialconcentrationofthetheseCtvaluesisplottedagainstthelogoftheinitialcopytargettemplateishigher,theCtwillbereachedatanearliernumber(Fig.2).ItshouldbeensuredthattheCtvalueoftheamplificationcycle.mostdilutedtemplateDNAusedtoconstructthestandardQuantificationoftheinitialtargetsequencesofancurveisatleastalogfoldlower(3.3cycles)thantheCtvalueunknownconcentrationisdeterminedfromtheCtvaluesofthenotemplatecontrol(NTC).Quantificationoftheandcanbedescribedeitherinrelativeorinabsoluteterms.unknowntargettemplateisdeterminedbycomparisonofInrelativequantification,changesintheunknowntargetaretheCtvaluesofthetargettemplateagainstthestandardexpressedrelativetoacoamplifiedsteadystate(typicallyacurve.However,inreality,thisabsolutequantificationofhousekeeping)gene.Anyvariationinthepresence(orthetargetgenerepresentsquantificationofthetargetinexpression)ofthehousekeepinggenecanpotentiallymaskcomparisonagainstaconstructedstandardcurve,ratherrealchangesorindicateartificialchangesintheabundancethanasanabsolutemeasurementofthenumberoftargetofthegeneofinterest.Whilethisapproachiscommonlygenespresentwithinanenvironmentalsample.Anynumberappliedforstudyingeukaryoticgeneexpression(reviewedoffactorsinvolvedintheconstructionofthestandardcurveinBustin,2002),itismoredifficulttoapplythismethodforincludingtheinitialquantificationofthestandardcurvestudyingprokaryoticgeneswheretheidentificationofatemplate,serialdilutionofthetemplateandthealgorithmicvalidsteady-statereferencegeneisproblematic.BurgmanndeterminationoftheCtvalue(Loveetal.,2006)contributeetal.(2007)neverthelesssuccessfullyutilizedsuchantothefinalquantificationoftheenvironmentalsample.Asaapproachwhenconfirmingmicroarray-baseddeterminationconsequence,itisrecommendedthatthefollowingdescrip-ofthetranscriptionalresponsesofSilicibacterpomeroyitotorsofthestandardcurvearereportedforeachQ-PCRdimethylsulphoniopropionateadditions.Frommicroarrayamplification:amplificationefficiency(E),thelinearregres-2experiments,theyidentifiedagenewhoseexpressionwassioncoefficient(r)andespeciallythey-interceptvalue,notalteredbyexperimentalconditionsandusedtheexpres-whichuniquelydescribesthestandardcurveandindicatessionofthisgenetonormalizelevelsofexpressionofthethesensitivityofthereaction(Smithetal.,2006;Fig.2).targetgenesofinterestinRT-Q-PCRassays.InanumberofFurthermore,theCtvalueoftheNTCanditsequivalentotherstudies,geneandtranscriptnumbersofthetargetgenevalueintermsofgenenumbersshouldbereported.More-ofinteresthavebeennormalizedtothenumbersof16Sover,wehavepreviouslydemonstratedthatevenhighlyrRNAgeneortranscripts(Neretinetal.,2003;Treuschetal.,reproduciblestandardcurvesmayresultinstatistically2005;Kandeleretal.,2006).Forexample,Treuschetal.significantdifferencesingenenumbersforthesametem-(2005)normalizedthenumberofamoAtranscriptstoplate(withequivalentCtvalues)whengenenumbersarenumbersof16SrRNAgenetranscriptsinRNAextractedquantifiedwithindifferentQ-PCRassays(Smithetal.,fromammonia-amendedorunamendedsoils.They2006)duetothelognatureofthecurve,wherebyminorreportedastatisticallysignificantincreaseinamoAtran-differencesinCtvaluesandstandardcurvesresultinlargescriptnumbersintheammonia-amendedsoils.However,differencesingenecopynumbers.although16SrRNAgenesandtranscriptsarenowcom-monlyusedinthismanner,theapplicationofsuchanBiologicalandmethodologicalfactorsapproachiscontroversial,especiallywhenstudyinggenes/affectingquantificationofgenesandtranscriptsamplifiedfromnucleicacidsextractedfromtranscriptsfromenvironmentalsamplescomplexenvironmentalsamples.Thisis,inparticular,because16SrRNAgenecopyandtranscriptnumbersareQ-PCR-basedquantificationofgene/transcriptnumbershighlyvariable,withthenumberof16SrRNAgenesperamplifiedfromnucleicacidsisolatedfromenvironmentaloperonvaryingdramaticallybetweenspecies(115copies)samplesisfurtherinfluencedbyanumberofothercom-while16SrRNAgenetranscriptionratesareregulatedpoundingfactors.Firstly,thechoiceofmethodusedforprimarilybyresourceavailability(Klappenbachetal.,nucleicacidextractionwillbeamajordeterminantonthe2000).The16SrRNAgenesandtranscriptscannotthereforefinalquantification.Nucleicacidextractionefficienciesvarybeconsideredasasteady-state(housekeeping)gene,espe-considerablybetweendifferentmethodsandthefinalciallywhenstudyinggenes/transcriptsinenvironmentalnucleicacidyieldisdependentonboththemethodusedsamples.andthetypeofenvironmentalsamplebeingstudiedInabsolutequantificationprotocols,thenumbersofa(Martin-Laurentetal.,2001).Moreover,manydifferenttargetgeneortranscriptaredeterminedfromastandardextractionprotocolsareusedfordifferentenvironmentalc2008FederationofEuropeanMicrobiologicalSocietiesFEMSMicrobiolEcol67(2009)6–20PublishedbyBlackwellPublishingLtd.Allrightsreserved ApplicationofQ-PCRinmicrobialecology11samplesandwithindifferentlaboratories,makingdirectcom-singleQ-PCRassayandusingthesamestandardcurveparisonofabsolutegenenumbersbetweenstudiesextremely(Smithetal.,2006).problematic.Hence,inordertocomparegene/transcriptnumbersfromdifferentenvironmentalsamples,itmustfirstQuantifyinggeneexpressionbyRT-Q-PCRbeensuredthatthesameextractionprocedureisusedforeachsample.Furthermore,whilethepresenceofPCRinhibitorsinCombiningQ-PCRwithaninitialRTreactionfacilitatesthenucleicacidsextractedfromenvironmentalsamplesandtheirquantificationofRNAtranscripts(rRNAormRNA),en-subsequenteffectonQ-PCRiswellestablished(Stultsetal.,ablingquantitativeestimatesoftheactivityofspecifictaxa2001),theconcentrationatwhichinhibitorsnolongeraffecttheorfunctionalguildswithinamicrobialcommunity.Isola-Q-PCRforanysampleisnotknownaprioriandmustbetionoftotalRNAdirectlyfromcomplexenvironmentaldeterminedempirically(Stultsetal.,2001)toensurethatthesamplesistypicallyproblematic,asRNAisalabilemoleculeenvironmentaltemplateandthestandardcurvetargetgenewithapotentiallyshorthalf-life(Grunberg-Manago,1999).haveequivalentamplificationefficiencies.AswithDNAquantification,thefirststeptowardsaccurateThesensitivityofQ-PCRallowsquantificationofveryRNAquantificationliesinthepreparationofahigh-qualitylownumbersoftargetgenes,withdetectionlimitsaslowastemplate,freefrominhibitors(Stultsetal.,2001).However,twocopiesofageneinaQ-PCR(Feyetal.,2004)reportedsimpledilutiontoreducetheconcentrationofinhibitorsintheliterature.However,statementspertainingtothepresentintheRNAtemplatemaybeproblematicasthesensitivityandlowerdetectionlimitsofQ-PCRshouldbekineticsoftheRTreactioncanbeaffectedadverselybylowqualifiedbyprovidinginformationontheamplificationRNAtemplateconcentrations(Chandleretal.,1998).Thissignaldetected,ifany,withintheNTC.ThisisbecauseeffecthasbeendemonstratedinenvironmentalsamplesquantificationoflownumbersofthetargetgenemaybeusingadilutionseriesofenvironmentalRNAwithintheRTartificiallyincreasedbythepresenceofanamplificationreaction,whichresultedinatleastlogfolddifferencesinthesignalwithinthereactionthatisequivalenttothatquanti-numberoftranscripts(transcriptspergramsediment)fiedwithintheNTC.WhiledetailsofamplificationsignalsquantifiedfromdifferentdilutionsofRNA(Smithetal.,intheNTCaresometimesreportedintheliterature(Suzuki2006).Moreover,duetothesensitivityof(fluorescence-etal.,2000;Gruntzigetal.,2001;Baldwinetal.,2003;Smithbased)Q-PCRmethods,itisparticularlyimportantthattheetal.,2006;McKewetal.,2007),manystudiesdonotRNAtemplateisfreefromcontaminatingDNAthatcouldprovidesuchdetails(Panickeretal.,2004;Kandeleretal.,contributetothefinalamplificationsignal.Absolutenum-2006;Coolenetal.,2008).Itis,however,recommendedthatbersofRNAtranscriptsshouldbedeterminedfromstan-detailsoftheCtvaluesoftheNTCandtheirequivalentgenedardcurvesconstructedfromcDNA(i.e.reversetranscribednumbersshouldbereportedforallQ-PCRassaysinordertoRNA)andnotfromadouble-strandedDNAtemplate.determinethelowerlimitsofdetectionforthereaction.ToFurthermore,theefficiencyoftheinitialRTstepiscriticalensurethattheNTC(ifdetected)doesnotcontributetotheforsensitiveandaccuratequantificationastheamountoffluorescencesignalofeitherthestandardcurveorthetargetcDNAproducedmustaccuratelyreflectthestartingRNAsequenceintheenvironmentalDNAsample,itisrecom-concentration.mendedthattheCtvalueofboththemostdiluteDNART-Q-PCRamplificationscanbeconductedusingeitherstandardandoftheunknowntargetgeneshouldhaveCtaone-steporatwo-stepreaction.Inaone-stepRT-Q-PCR,valuesof3.3cycles(alogvalue)fewerthanthatoftheNTCboththeRTreactionandtheQ-PCRarecarriedoutCtvalue(Smithetal.,2006).consecutivelyinasingletube.RNAisfirstreversetran-Thedeterminationofgeneandtranscriptnumbersam-scribed,withallresultantcDNAservingastemplatesintheplifiedfromenvironmentalsamplesgeneratedbydifferentsubsequentQ-PCRamplification.Inadditiontotheresearchgroupswillentailanynumberoftheaforemen-reducedriskofcontaminationandtheconvenienceoftionedvariablesintheQ-PCRprotocolandmaybeaffectedsettinguponlyasinglereaction,afurtheradvantageofthisbytheinitialextractionofnucleicacids,thepreparation,methodisthatalltheresultingcDNAproducedisusedtoquantificationandamplificationofthestandardcurvequantifythetargetRNAsequence.However,forthestudyoftemplate(Loveetal.,2006),variationsintheefficienciesofeukaryotes,one-stepRT-Q-PCRreactionshavebeenre-thesubsequentQ-PCR,differencesintheQ-PCRplatform,portedtohavereducedsensitivity(Bustin,2002)asreactionassociatedsoftwareandreagentsthatareused,aswellasconditionsarecompromisedtoaccommodatethetwovariationsduetodifferentresearchersandlaboratories.differentenzymesrequiredwithinasinglereaction.InaTherefore,thegenerationofabsolutegenenumberscantwo-stepRT-Q-PCRprotocol,theRTreactionandtheonlybeconsideredasbeingmeaningfulfortheindividualsubsequentQ-PCRarecarriedoutseparately.Firstly,cDNAstudyinquestionandeventhen,suchadirectcomparisonisgeneratedinanindependentRTreactionandsubse-shouldbeusedonlyforgenenumbersdeterminedwithinaquentlyanaliquotofthiscDNAisusedasatemplateforFEMSMicrobiolEcol67(2009)6–20c2008FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.Allrightsreserved 12C.J.Smith&A.M.OsborntheQ-PCR.AnadvantageofthismethodisthattheRTmizationtoensureasinglespecificampliconandthatreactioncanbeoptimizedtoincreasecDNAyieldascantheprimerdimersarenotproduced;thismustalsobecon-subsequentQ-PCRamplification.Furthermore,cDNAgen-firmedbydissociationcurveanalysis.HighlyreproducibleeratedintheRTreactioncanbeusedasatemplateforaDNAandRNAstandardcurvescanbecreatedbydilutionofnumberofdifferentQ-PCRreactions.Ifrandomprimersknownconcentrationsofstandards(seeSmithetal.,2006areusedintheinitialRTreaction,thenanynumberoffordetails).Careshouldbetakentoavoidrepeatedfree-subsequentgene-specificQ-PCRamplificationsusingthezethawingoftemplatesusedtoconstructstandardcurves.randomlyprimedcDNAcanbeconducted,makingatwo-Asquantificationofgenesortranscriptsfromanenviron-stepreactionamoreeconomicallyviableoptionformentalsamplesiscalculatedfromthestandardcurve,afull2RT-Q-PCRthanaone-stepreaction.Whilerandomprimersdescriptionofthestandardcurve(r,slope,efficiencyandcanmaximizethenumberofdifferentcDNAtemplatesy-interceptvalue)shouldbegivenwhenreportinggeneand/generated,gene-specificreverseprimerscanincreasetheortranscriptnumbers.Biological(notjusttechnical)repli-sensitivityandspecificityofthecDNAcreatedandatthecation(atleastn=3)isessentialfor(RT)-Q-PCRtoenablesametimereducetheamountofunspecificbackgroundstatisticalinvestigationofdifferencesingeneortranscriptcDNA.However,thismaybedependentonthegene-specificnumbersbetweensamplesortreatments.Finally,asthereareprimer.Forexample,N´ıcolaisenetal.(2008),inastudyofnumerouscompoundingfactorsthatcanaffectquantifica-tfdAgeneexpressioninsoil,showedthatuseofrandomtion,werecommendthatcomparisonsbetweenabsoluteprimersintheRTreactionasopposedtousinggene-specificgeneortranscriptnumbersgeneratedfromdifferentQ-PCRprimerswasoptimalforRT-Q-PCRoftfdAtranscripts.assays(orindeedstudies)shouldnotbemade.Clearly,thechoiceoftheprotocol,enzyme,primerandreactionconditionswillinfluencethequantificationoftheApplicationofQ-PCRforinvestigatingtheRNAtemplatefromtheenvironment.microbialgeneticpotentialwithintheenvironmentPracticalitiesof(RT)-Q-PCRprotocolsThefirstapplicationsofQ-PCRinmicrobialecologywereAlthoughthephysicalset-upof(RT)-Q-PCRamplificationsreportedinthreepaperspublishedinNovember2000,toquantifygeneortranscriptnumbersfromenvironmentalwhichusedTaqMan-basedassaystotarget16SrRNAgenesnucleicacidsisstraightforward,thequantitativedatagener-(Beckeretal.,2000;Suzukietal.,2000;Takai&Horikoshi,atedfromthesereactionscanbeaffectedbymanycom-2000).Beckeretal.(2000)demonstratedtheabilityofpoundingfactors.Consequently,suchfactorsasdiscussedTaqManprobestodeterminetheabundanceofaspecificearlierinthisreviewneedtobecarefullyconsideredwhenecotypeofSynechococcussp.BO8807againstamixeddesigning,developingandimplementing(RT)-Q-PCRpro-backgroundofphylogeneticallyrelatedbacteriausingartifi-tocols.Detailsofsomeadditionalkeyconsiderationsandcialmixedcommunities.Suzukietal.(2000)exploitedtherecommendationsfortheuseof(RT)-Q-PCRaregivenspecificityandthesensitivityofTaqManQ-PCRassaystobelow.determinespatialandtemporalquantitativedifferencesin(RT)-Q-PCRampliconsshouldbeshort,ideallybetweenthedistributionsofSynechococcus,Prochlorococcusandar-50and150bpinlength.While,theGCcontentofthechaeainmarinewaters,whileTakai&Horikoshi(2000)primerscanrangebetween20%and80%(althoughpairedquantifiedarchaeal16SrRNAgenenumberswithinsamplesprimersshouldhavesimilarmeltingtemperatures;Tm),afromadeepseahydrothermalventeffluent,hotspringwaterhighGCcontentwillincreasethespecificityofthereaction,andfromhotspringandfreshwatersediments.BytargetingwhichisofparticularimportanceforSYBRgreenassays.highlyconservedregionsofthe16SrRNAgene,Q-PCRWhendesigningaTaqManprobe,theprobeshouldbeassayshavebeendesignedtoquantifytotalbacterial(and/situatedascloseaspossibletotheforwardprimerwithoutorarchaeal)numberswhiletargetingoftaxa-specificoverlapping.Theprobeshouldnothaveaguaninenucleo-sequenceswithinhypervariableregionswithinthegenetideatthe50endorhavemoreguaninesthancytosinesasenablesquantificationofsequencesfromphylumtospeciesguanineresiduesarenaturalquenchers.TheTmoftheprobelevels,providedthattherearesequencedataavailablethatshouldbe8101CabovetheTmoftheprimers.Whenenablethedesignofprimersandprobes.Acaveatofthisdesigningprimersandprobes,itmaybedifficulttomeetapproachmustbestressed;16SrRNAgenenumbersfromalltheabovecriteria.However,satisfyingasmanyoftheseasenvironmentalsamplescannotbeconvertedtocellnumberspossiblewithintheconstraintsoftheassaydesignwillastheexactnumberofcopiesofthe16SrRNAgeneinanymaximizethelikelihoodofsuccessfulquantification.Asgivenbacterialspeciesvaries(Klappenbachetal.,2000).outlinedearlier(seeFluorescencedetectionchemistrysec-Table1detailscommonlyusedrRNAQ-PCRprimerandtion),SYBRgreenprimersetsmayrequireextensiveopti-probesets.c2008FederationofEuropeanMicrobiologicalSocietiesFEMSMicrobiolEcol67(2009)6–20PublishedbyBlackwellPublishingLtd.Allrightsreserved ApplicationofQ-PCRinmicrobialecology13Table1.QuantitativePCRprimerandprobesetstargetingsmallsubunitribosomalRNAgenesofbacteria,archaeaandfungiDetectionAmpliconTemp.TargetchemistryPrimer/probeSequence(50–30)length(bp)(1C)ReferencesProkaryoteTMUni340FCCTACGGGRBGCASCAG46657Takai&Horikoshi(2000)16SrRNAgeneUni806RGGACTACNNGGGTATCTAATTM516FTGYCAGCMGCCGCGGTAAHACVNRSBacterialTMBACT1369FCGGTGAATACGTTCYCGG12356Suzukietal.(2000)16SrRNAgenePROK1492RGGWTACCTTGTTACGACTTProbeTM1389FCTTGTACACACCGCCCGBacterialTM331FTTCTACGGGAGGCAGCAG46660Nadkarnietal.(2002)16SrRNAgene797RGGACTACCAGGGTATCTAATCCTGTTProbeBacTaqCGTATTACCGCGGCTGCTGGCACArchaealTMArch349FGYGCASCAGKCGMGAAW45759Takai&Horikoshi(2000)16SrRNAgeneArch806RGGACTACVSGGGTATCTAATTMArch516FTGYCAGCCGCCGCGGTAAHACCVGCArchaealSGAr109fACKGCTCAGTAACACGT80652Lueders&Friedrich(2003)16SrRNAgeneAr915rGTGCTCCCCCGCCAATTCCTFungalSGEUK345FAAGGAAGGCAGCAGGCG14960Zhuetal.(2005)18SrRNAgeneEUK499RCACCAGACTTGCCCTCYAATFungalSGFung5fGTAAAAGTCCTGGTTCCCC55048Smitetal.(1999)18SrRNAgeneFF390rCGATAACGAACGAGACCTVainio&Hantula(2000)Luedersetal.(2004)TM,TaqManprobe;SG,SYBRgreen;Temp.,annealingtemperature.Quantificationofeukaryoteswithinenvironmentalsam-etal.,2007),nitratereductionanddenitrification(Lopez-plesbyQ-PCRcanbecarriedoutbytargetingthe18SrRNAGutierrez´etal.,2004;Henryetal.,2006;Smithetal.,2007),gene(Luedersetal.,2004;Zhuetal.,2005)ortheinternalsulphatereduction(Leloupetal.,2007),methanogenesistranscribedspacer(ITS)region(Landeweertetal.,2003;(Denmanetal.,2007)andmethaneoxidation(Kolbetal.,Kennedyetal.,2007).TheITSregionisoftentargetedfor2003)havebeeninvestigated(seeTable2fordetailsofthedesignoftaxon-specificQ-PCRassaysasitprovidesanitrogencycleQ-PCRanalyses).InaparticularlystrikinggreaterdegreeofsequencedifferentiationbetweenspeciesexampleofthevalueofsuchfunctionalgeneQ-PCRassays,andlowerwithin-speciesvariability(Kennedyetal.,2007)therelativecontributionsofammonia-oxidizingarchaeathanisseenforthe18SrRNAgene.Aswithquantificationofandbacteriatothefirststepofnitrification(ammonia16SrRNAgenenumbers,Q-PCR-derivedITSregionandoxidation)havebeeninvestigatedbothinsoils(Leininger18SrRNAgenenumberscannotbedirectlyequatedtocelletal.,2006;Heetal.,2007b)andinseawater(Minceretal.,numbers.However,numbersoffungalrRNAgeneorITS2007)bydeterminationoftheabundanceofarchaeal-andnumberspervolumeofsamplecanbeusedtocomparethebacterial-relatedamoAgenes.Thesestudieshavesuggestedrelativenumbersoffungibetweendifferentenvironmentalthatarchaeaandnotbacteriaarethenumericallydominantsamples(Guidotetal.,2002).ammoniaoxidizersinbothenvironments.TheresultsofInadditiontoquantitativedataontaxonomicmarkers,suchstudiesarethereforeencouragingare-evaluationofourQ-PCRhasalsobeenappliedtoquantifyfunctionalgenesbasicunderstandingofnitrogencyclingandtherelativewithintheenvironment.Bytargetingfunctionalgenesthatimportanceofbacteriaandarchaea(orspecifictaxaorencodeenzymesinkeymetabolicorcatabolicpathways,thefunctionalguildswithinthedomains)withinkeyenviron-(genetic)potentialforaparticularmicrobialfunctionwith-mentalprocesses.Whilethesestudieshavegreatlyenhancedinaparticularenvironmentcanbeassessed.Tounderstandourunderstandingofgenenumbersintheenvironment,themicrobialfunctioningintheenvironmentatamolecularnextsteptofurtherourunderstandingistolinkvariationinlevel,itisessentialnotonlytoknowwhatgenesarepresentgeneticpotential(i.e.genenumbers)withinasysteminandthediversityofthesegenesbutalsotodeterminetheirrelationtovariationinratesandactivityofthebiologicallyabundanceanddistributionwithintheenvironment.Tothisdrivenenvironmentalprocessesinquestion,andhenceend,Q-PCRassayshavebeendesignedtotargetmicrobiallyenablingimprovedunderstandingoftheunderpinningmediatedbiogeochemicalprocessesintheenvironment.factorsthatinfluencemicrobialfunctioningwithintheQuantificationoffunctionalgenesinvolvedinammoniaenvironment.AsQ-PCRisasensitiveandspecificmethodoxidation(Hermansson&Lindgren,2001;Okanoetal.,totrackchangesintheabundance(andexpression)of2004;Treuschetal.,2005;Leiningeretal.,2006;Mincerspecifictargetfunctionalgenes,itlendsitselftoexperimentsFEMSMicrobiolEcol67(2009)6–20c2008FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.Allrightsreserved 14C.J.Smith&A.M.OsbornTable2.Q-PCRprimersandprobestargetinggenesencodingenzymesinvolvedinnitrogencyclingFunctionalgroupTargetgeneComment/environmentReferencesNitrogenfixationnifHSuiteofTaqManprobesandprimersdesignedtoquantifynifHtranscriptsChurchetal.(2005)fromseawaterSYBRgreenprimerstargetingthenifHgeneofSynechococcussp.OS-B’Steunouetal.(2006)isolateandusedtoquantifynifHtranscriptsfromahotspringmicrobialmatAmmoniaoxidationamoATaqmanprobeandprimerstargetingknownbacterialammoniaoxidizers.Okanoetal.(2004)UsedtoquantifygenesfromsoilTaqManprobeandprimersdesignedfromthealignmentofenvironmentalTreuschetal.(2005)mRNAandDNAclonesfromsoilsamples.UsedtoquantifytranscriptsfromasoilmicrocosmNitratereductionnarGSYBRgreenprimersdesignedfromenvironmentalsoilclonelibraries.UsedLopez-Gutierrez´etal.(2004)toquantifynarGfromarangeofsoiltypesSuiteofTaqManprimersandprobesdesignedfromenvironmentalcloneSmithetal.(2007)library.UsedtotargetnarGgenesandtranscriptsfromestuarinesedimentsSYBRgreenprimersetdesignedfromallavailablenarGsequencesintheBruetal.(2007)publicdatabaseandusedtoquantifygenesfromriversediment,rangeofsoils,waterandbiofilmsnapASuiteofTaqManprimersandprobesusedtotargetnapAgenesandSmithetal.(2007)transcriptsfromestuarinesedimentsSYBRgreenprimersetusedtoquantifygenesfromriversediment,soils,Bruetal.(2007)waterandbiofilmsNitritereductionnirSTaqManprobesandprimerstargetingPseudomonasstutzeri-relatednirSGruntzigetal.(2001)genes.UsedtoquantifynirSgenesfromsoilandcontaminatedgroundwaterSuiteofTaqManprimersandprobesdesignedfromnirSmRNAcloneSmithetal.(2007)library.UsedtoquantifygenesandtranscriptsfromestuarinesedimentsnirKSYBRgreenprimersetdesignedfromallnirKsequencesavailableattheHenryetal.(2006)time.UsedtoquantifygenesfromarangeofsoiltypesNitricoxidereductionnorBTwoSYBRgreenprimersetstargetingthecytochromecelectrondonorDandieetal.(2007)(cNOR)formoftheenzyme;designedfromculturedsoilisolates.UsedtoquantifycnorBfromsoilmicrocosmsNitrousoxidereductionnosZTwoSYBRgreenprimersetsdesignedfromdiversenosZsequences.UsedHenryetal.(2006)toquantifynosZgenesfromarangeofsoiltypesNitrateammonificationnrfATaqManprimersandprobetargetingnrfAinestuarinesedimentsSmithetal.(2007)thatfurtherinvestigatetheenvironmentalcontrols/effectsFunctionalgenesencodingkeyreactionsinbiodegrada-onthenumbersofthetargetgene(andhencetheorganismstionpathwaysofenvironmentalpollutantshavealsobeencarryingthesegenes)andsubsequentlyontheenvironmen-targetedbyQ-PCRanalysis(Baldwinetal.,2003,2008;talprocessthatthesegenes(andorganisms)encode.ADeversetal.,2004;Gonodetal.,2006;McKewetal.,2007;recentstudybyDandieetal.(2007)hasadoptedsuchanseeTable3fordetailsofQ-PCRprimerand/orprobesets).approachbyquantifyingtheresponseofdenitrifyingpopu-Theaccuratequantificationofkeygenessuchasthoselationswithinsoilmicrocosmsamendedwithvaryingcon-encodingmono-oxygenaseanddiooxygenaseenzymesin-centrationsofglucose(asanelectrondonor)designedtovolvedinthecatabolicconversionsofenvironmentalpollu-inducedifferentratesofdenitrification.Denitrifierpopula-tantsinsituwillgreatlyenhanceourunderstandingandtionnumberswereassessedusingthenitricoxidereductaseimportantlyimproveourknowledgeofthebioticpotential(cnorB)geneasaproxyfordenitrifiernumberstargetingwithinanenvironmentforsuccessfulbioremediation,andtwopopulations(cnorBP:PseudomonasandcnorBB:Bosea,furtherhowindigenousoraugmentedmicroorganismsBradyrhizobium,Ensifer).Thesemesocosmexperimentsin-respondtobiostimulationprotocols.Forexample,thedicatedthatdenitrificationratesandmicrobialrespirationeffectsofbiostimulationandbioaugmentationremediationincreasedsignificantlywithincreasingadditionofglucosestrategiesontheactivityofhydrocarbondegradingbacteriaandthatthiswasaccompaniedbyincreasesincnorBP,butinseawatermicrocosmscontainingcrudeoilwereinvesti-notcnorBBpopulations,revealingpopulation-specificgatedovera30-dayperiodbytargetingalkanehydroxylaseresponsestocarbonamendment.andaromaticringhydroxylatingdioxygenasegenesbyc2008FederationofEuropeanMicrobiologicalSocietiesFEMSMicrobiolEcol67(2009)6–20PublishedbyBlackwellPublishingLtd.Allrightsreserved ApplicationofQ-PCRinmicrobialecology15Table3.Q-PCRprimersandprobestargetinggenesinvolvedinbiodegradationTargetchemicalFunctionalgroupTargetgeneComment/environmentReferencesHerbicideatrazineAtrazinedegradingbacteriaatzA,B,C,EandFSYBRgreenprimerstargetingatzDeversetal.(2004)catabolicgeneexpressionintwoatrazine-degradingbacteriaHerbicideMCPAMCPA(4-chloro-2-tfdASYBRgreenprimerstargetingtfdAVallaeysetal.(1996)methylphenoxy-aceticacid)geneinsoil.Primersdescribedoriginally(primers)Gonodetal.and2,4-dichlorophenoxyaceticbyVallaeysandcolleagues,andlater(2006)(Q-PCRassaydegradingbacteriaadaptedbyGonodandcolleagues,conditions)forQ-PCRSYBRgreenQ-PCRprimersetdesignedBælumetal.(2006)fromthealignmentof23knowntfdAgenesandusedtotrackquantitativechangesintfdAgenenumbersinsoilduringdegradationofMCPATrichloroetheneandBacteriainvolvedin16SrRNAgeneSuiteofgroupspecificprimersforSYBRMilleretal.(2007)cis-dichloroethenereductivedechlorinationgreenQ-PCRtargetingofCFB,(cis-DCE)ofTCE2andoxidationofAlphaproteobacteriaandBurkholderialescis-DCEinhydrocarbon,trichloroetheneandcis-DCEcontaminatedgroundwaterChlorinatedethenesAnaerobicreductivetceA,vcrAandbvcATaqmanprimerandprobesetstargetingLeeetal.(2008)dehalogenases(RDase)thetceA,vcrAandbvcAgenesofDehalococcoidesspp.ingroundwaterHalogenatedcompoundsReductivedehalogenating16SrRNAandrdhSYBRgreenprimersetstargetingtheAhnetal.(2007)bacteriagenes16SrRNAgeneoftwoknowndehalogenatingbacteriaandaSYBRgreenprimersettargetingardhgenedesignedfromsequencesretrievedfrommarinesedimentsamendedwith1,2,3,4-tetrachlorodibenzo-p-dioxin(TeCDD)Methyltert-butyletherMTBE-degradingbacterial16SrRNAgeneTaqmanprimerandprobesettargetingHristovaetal.(2001)(MTBE)strainPM1theMTBEdegradingbacterialstrainPM1ingroundwaterandsedimentsHydrocarbons(aliphaticHydrocarbonoclasticalkB2,alkB,phnASYBRgreenprimerstargetingthealkaneMcKewetal.(2007)andaromatic)bacteriahydroxylaseinAlcanivoraxborkumensisandThakassolituusoleivoransandthearomaticring-hydroxylatingdioxygenasegenefromCycloclasticusspp.Hydrocarbons(aromatic)Toluene-andxylene-degradingbssATaqmanprobeandprimersettargetingBelleretal.(2002)bacteriabssAgeneinavarietyoftoluene-degradingdenitrifyingbacteriaHydrocarbons(aromatic)AromaticcompounddegradingEntiresubfamiliesofPaperoutlinesthedevelopmentofaBaldwinetal.(2003)bacteriarelatedoxygenasesuiteofSYBRgreenprimersetstargetinggenesratherthanbiphenyldioxygenase,naphthalenespecies-specificdioxygenase,toluenedioxygenase,genestoluene/xylenemonooxygenases,phenolmonooxygenaseandring-hydroxylatingtoluenemonooxygenasegenesQ-PCRwhilesimultaneouslymeasuringthedegradationofbiodegradationofotherpollutants,suchasherbicides.Forthecrudeoil(McKewetal.,2007).Thisstudyrevealedthatexample,Q-PCRhasbeenusedtostudythepotentialforspecifictaxawithinthesehydrocarbon-degradingbacterialbiodegradationoftheherbicide4-chloro-2-methylphenox-communitiesweredirectlyinfluencedbyapplicationofyaceticacid(MCPA)indifferentsoiltypes(Bælumetal.,differentbiostimulationapproachesinvolvingadditionof2006)bytargetingandquantifyingthetfdAgeneinvolvedinnutrientsand/orbioemulsifiers.Q-PCRisavaluabletoolfortheinitialdegradationstepofthecompound.Thisstudyinvestigatingthepotentialwithintheenvironmentforshowedafive-andthreefoldlogincreaseintfdAgeneFEMSMicrobiolEcol67(2009)6–20c2008FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.Allrightsreserved 16C.J.Smith&A.M.OsbornnumbersovertimeinsoilmicrocosmsamendedwitheitherRT-Q-PCRtoquantifyexpressionofreductivedehalogenase11ahigh(20mgkg)oralow(2.3mgkg)doseofMCPA,(vcrA,bvcAandtceA)genesasbiomarkersofDehalococcoidesrespectively,withincreasesintfdAgenesinverselypropor-spp.activityandtodistinguishtherolesofdifferentstrainsoftionaltoMCPAdegradation.Moreover,thisstudyalsoDehalococcoidesduringbioremediationandbioaugmentationdemonstratedthediagnosticpotentialofusingSYBRgreenofgroundwatercontaminatedwithtrichloroethene.RT-Q-dissociationcurveanalysisofQ-PCRproductstoidentifyPCRindicatedthatvcrAandbvcAgenetranscriptswerehighlyshiftsinthedominanttfdApopulationsovertimeandexpressedinallsamples,whereasthetceAtranscriptswereduringdegradation.Subsequentclonelibraryanalysisinconsistentlyquantifiedandwereatlowerlevels,indicatingshowedthatclassIIItfdAgeneswereresponsibleforMCPAthatDehalococcoidesspp.carryingvcrAandbvcAgenesplayeddegradationandnotclassItfdAgenes,whichweredomi-amoreimportantroleintrichloroetheneinsitubioremedia-nantbeforethedegradationprocesswasinitiated.tion.ThesetwoexampleshighlighthowtheapplicationofRT-Q-PCRintheenvironmentwillundoubtedlyfurtherourQuantifyinggeneexpressioninunderstandingofthemanyimportantprocessesthatareenvironmentalsamplesusingRT-Q-PCR:amediatedbymicroorganisms.stepclosertodeterminingthefunctioningoftargetgenesintheCombining(RT)-Q-PCRwithotherenvironmentapproachestoprovidegreaterinsightintocommunityfunctionanddynamicsRT-Q-PCRcanbeusedtodetectandquantifymRNAtranscriptsofinterestincomplexenvironmentalsamplesLinkingthestructureandcompositionofmicrobialcom-bothinasensitiveandaspecificmanner.However,munitieswiththebiologicalfunctionthatindividualspeciesRT-Q-PCRtoinvestigategeneexpression(rRNAormRNA)orfunctionalguildsconveyisakeyobjectivewithinmicro-withinenvironmentalsampleshasbeenfarlesswidelybialecology.Stableisotopeprobing(SIP)(Radajewskietal.,appliedthanQ-PCR-basedassessmentofgenenumbers2000;Manefieldetal.,2002)canbeusedtodirectlylink(i.e.fromDNA)intheenvironment.ThisisprimarilyduedistincttaxawithinamixedmicrobialcommunitytotothedifficultiesofextractingintactRNA,andparticularlyspecificmetabolicprocesses,particularlycarbonutilization/13intactmRNA,fromenvironmentalsamples.Whilethedegradation,usinglabelledsubstratessuchasC.DuringquantificationofbothrRNAgenesand/orfunctionalgenesmicrobialgrowth,thesesubstratesareincorporatedintothefromtheenvironmentcanbeusedasanindicatorofthenucleicacids(DNAorRNA)frommembersofthecommu-geneticpotentialwithinanenvironmentandissuggestiveofnitythataredirectly(orindirectly)utilizingthelabelledpotentialfunctionalactivitywithinacommunity,molecularsubstrate,andtheheavylabellednucleicacidcanbeinvestigationofbiologicalactivityshouldpreferablydeter-separatedusingdensitygradientultracentrifugationfromminechangesingeneexpressionandideallyofmRNAthe(unlabelled)nucleicacidsrepresentativeofothermem-transcriptsencodedbyspecificfunctionalgenes.Alimitedbersofthecommunitythatdonotutilizethesubstrate.numberofstudieshaveindeedshownthesuccessfulquanti-Luedersetal.(2004)combinedSIPwith(RT)-Q-PCRforficationbyRT-Q-PCRofanumberoffunctionalgenequantitativemeasurementsofadomain-specifictemplatetranscriptsfromarangeofenvironmentsincludingaquaticdistributionthroughthedifferentiallylabelledfractionsofecosystems(Holtzendorffetal.,2002;Wawriketal.,2002;DNAandRNAextractedfromsoilmicrocosmsfollowingFeyetal.,2004;Churchetal.,2005;Gonzalez-Escalonaetal.,SIPincubation.Thisenabledthecommunitydynamicsof2006;Leeetal.,2008)butalsoinestuarinesediments(Smithmethanotrophsinricefieldsoilstobetrackedovertime.etal.,2007),soil(Treuschetal.,2005;N´ıcolaisenetal.,Bacterial,archaealandeukaryoterRNAgeneswerequanti-2008),hotspringmicrobialmats(Steunouetal.,2006)andfiedintheheavylabelledfractions,indicatingnotonlythebloodandfaecalsamples(Matsudaetal.,2007).InapresenceofadynamicmethanotrophcommunitythatwasparticularlyelegantapplicationofRT-Q-PCR,Steunouenrichedovertimebutalsodirectorindirectincorporation13etal.(2006)investigatedchangesinexpressionofSynecho-ofthelabelledCintoeukaryotes(fungiandprotozoa).coccusspp.nif(nifH,nifD,nifK)genesinahotspringMicroarraysarenowbeingincreasinglyusedtosimulta-microbialmatovera12-hperiod.Transcriptsinthematneouslyscreenmicrobialcommunitieswithindiverseenvir-wereonlydetectedandquantifiedattheendoftheday,onmentsforthepresence(and,inprinciple,theabundance)whenthematbecameanoxic.Theyfurtherquantifiedofspecificribo-orphylotypes(taxa)and/orfunctionalexpressionofkeygenesinvolvedinphotosynthesis,respira-genes.MicroarrayplatformssuchasthePhyloChip(Brodietionandfermentationprocesseswithinthehotspringetal.,2007)andGeoChip(Heetal.,2007a)arenowmicrobialmattobuildanoverviewoftheenergy-generatingaffordingapreviouslyunparalleledopportunitytounder-processesthatmaydriveN2fixation.Leeetal.(2008)usedtaketargetedphylogeneticmarker-basedandfunctionalc2008FederationofEuropeanMicrobiologicalSocietiesFEMSMicrobiolEcol67(2009)6–20PublishedbyBlackwellPublishingLtd.Allrightsreserved ApplicationofQ-PCRinmicrobialecology17genesurveysofenvironments.Nevertheless,thepotentialSoginetal.,2006),suchdatasetsprovideonlysemi-forprovidingquantitativeassessmentsofgeneabundancequantitativeassessmentsofthediversityand/ortheabun-frommicroarraysasappliedtonucleicacidsextractedfromdanceofparticularphylotypesthatcanagainbevalidatedbyenvironmentalsamplesisoftencompromisedbythere-(RT)-Q-PCR-basedapproaches.quirementforaninitialamplificationstepfromtheenvir-onmentalDNA(orRNA),oftenviaPCRamplificationConclusions(Brodieetal.,2007),butalternativelyviarollinglinearInconclusion,(RT)-Q-PCR-basedapproachesrepresentamplification(Heetal.,2007a),beforemicroarrayhybridi-fast,effectivemethodsenablingthequantificationofgenezation.Consequently,andespeciallyinthosemicroarrayand/ortranscriptnumberswithinenvironmentalsamples,studiesrequiringanintermediatePCRamplification,anyprovidingunparalleledspecificityandsensitivitytotargetquantitativeinterpretationofsuchdatasetsshouldbesequencespresentwithinamixedcommunitybackground.treatedwithcaution,astheseresultswillbesusceptibletoAswithallmethodologies,thevalidityoftheresultingdatathesamebiasesthatareassociatedwithanyend-pointPCRsetsshouldbeconsideredagainstthespecificityandexperi-protocol(Reysenbachetal.,1992).Nevertheless,microarraymentalvariabilityassociatedwiththemethod.Inparticular,experimentscanbeusedtoidentifypotentiallyinterestingforQ-PCR-basedassays,thevalueofsuchdatasetsshouldquantitativechangesintaxon-orgene-specificabundancebeconsideredinrelationtothespecificityoftheprimer(andbetweenenvironmentalsamplesthatcanthenbevalidatedprobes)usedintheamplificationandwithrespecttobyQ-PCR-basedapproaches,andhenceQ-PCRcanbeinstrument,userandmostimportantlyexperimentalvaria-recommendedasafast,target-specificmethodforvalida-bilityassociatedwiththemethod.Moreover,inordertotionofthe(semi-)quantitativeresultsgeneratedfromthemaximizethevalueof(RT)-Q-PCR-basedapproachesforincreasingnumberofenvironmentalmicroarrays(Rheefurtheringbiologicalunderstandinginmicrobialecology,etal.,2004;Brodieetal.,2006,2007;Burgmannetal.,theirvalueisgreatestwhenusedincombinationwithother2007;Heetal.,2007a).(andoftenprocess-based)assessmentsofecosystemfunc-OnemajordisadvantageofQ-PCR-basedapproachesistion.therequirementforpriorsequencedataofthespecifictargetgeneofinterest.Consequently,Q-PCRcanonlybeusedfortargetingofknowngenes.Historically,anduntilrecently,Acknowledgementssequenceinformationhasprimarilybeenderivedfromgenomeorgenefragmentsequencesfromculturedorgan-TheauthorswouldliketothankJimProsserfortheismsand/orfromclonelibrariesgeneratedbyPCRusinginvitationtowritethisreview.ThisresearchwassupportedprimersthatarethemselvesbasedoncurrentsequencebyagrantawardedtoA.M.O.andC.J.S.fromtheUnitedknowledge.Hence,accessingtheunknownusingQ-PCRKingdomNaturalEnvironmentResearchCouncil(Ref.orindeedanyPCR-basedmethodsisinevitablylimitedtoNER/D103089/1).theanalysisofsequencesrelatedtothosethathavealreadybeencharacterized.Becausemolecularanalysisofenviron-Referencesmentalmicroorganismshasrepeatedlyshownthatthemajorityofmicroorganisms(andtheirgenes)intheenvir-AhnYB,HaggblomMM&KerkhofLJ(2007)Comparisonofonmentarehighlydivergentfromthoseofmostculturedanaerobicmicrobialcommunitiesfromestuarinesedimentsorganisms,thisrepresentsaCatch22situationfortheamendedwithhalogenatedcompoundstoenhancedevelopmentofnewPCR-basedassays.Inrecentyears,thisdechlorinationof1,2,3,4-tetrachlorodibenzo-p-dioxin.FEMSMicrobiolEcol61:362371.problemhas,however,beencircumventedbytheintroduc-BælumJ,HenriksenT,HansenHCB&JacobsenCS(2006)tionofmetagenomicapproachesthatprovideaPCR-Degradationof4-chloro-2-methylphenoxyaceticacidintop-independentassessmentofmicrobialdiversity.Twomainandsub-soilisquantitativelylinkedtotheclassIIItfdAgene.strategieshavebeenutilized,namelyclonelibrary-basedApplEnvironMicrob72:14761486.metagenomes(Verginetal.,1998;Bejaetal.,2000;VenterBaldwinBR,NakatsuCH&NiesL(2003)Detectionandetal.,2004)andmorerecentlyultra-high-throughputenumerationofaromaticoxygenasegenesbymultiplexandsequencingapproachessuchaspyrosequencing(Edwardsreal-timePCR.ApplEnvironMicrob69:33503358.etal.,2006;Dinsdaleetal.,2008).Thelatter,particularly,BaldwinBR,NakatsuCH&NiesL(2008)Enumerationofoffersconsiderablebenefitsbothintermsofprovidingmucharomaticoxygenasegenestoevaluatemonitorednaturallargerdatasetsthancanbegeneratedvialibrary-basedattenuationatgasoline-contaminatedsites.WaterRes42:approaches,andasimportantly,byavoidingpotential723731.sequence-specificcloningbiases.Moreover,wherepyrose-BeckerS,BogerP,OehlmannR&ErnstA(2000)PCRbiasinquencingisusedtotarget-specificgenes(e.g.rRNAgenes;ecologicalanalysis:acasestudyforquantitativeTaqnucleaseFEMSMicrobiolEcol67(2009)6–20c2008FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.Allrightsreserved 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