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Realtimepolymerasechainreaction
FromWikipedia,thefreeencyclopedia

Arealtimepolymerasechainreactionisalaboratory
techniqueofmolecularbiologybasedonthepolymerasechain
reaction(PCR).Itmonitorstheamplificationofatargeted
DNAmoleculeduringthePCR,i.e.inrealtime,andnotatits
end,asinconventionalPCR.RealtimePCRcanbeused
quantitatively(QuantitativerealtimePCR),semi
quantitatively,i.e.above/belowacertainamountofDNA
molecules(SemiquantitativerealtimePCR)orqualitatively
(QualitativerealtimePCR).

SYBRGreenfluorescencechartproducedinreal
timePCR.

TwocommonmethodsforthedetectionofPCRproductsin
realtimePCRare:(1)nonspecificfluorescentdyesthat
intercalatewithanydoublestrandedDNA,and(2)sequence
specificDNAprobesconsistingofoligonucleotidesthatare
labelledwithafluorescentreporterwhichpermitsdetection
onlyafterhybridizationoftheprobewithitscomplementary
sequence.
TheMinimumInformationforPublicationofQuantitative
RealTimePCRExperiments(MIQE)guidelinesproposethat
Meltingcurveproducedattheendofrealtime
theabbreviationqPCRbeusedforquantitativerealtimePCR
PCR.
andthatRTqPCRbeusedforreversetranscriptionqPCR.[1]
Theacronym"RTPCR"commonlydenotesreverse
transcriptionpolymerasechainreactionandnotrealtimePCR,butnotallauthorsadheretothisconvention.[2]

Contents
1 Background
2 Basicprinciples
3 Classification
3.1 RealtimePCRwithdoublestrandedDNAbindingdyesasreporters
3.2 Fluorescentreporterprobemethod
4 Fusiontemperatureanalysis
4.1 Quantificationofgeneexpression
4.2 Modeling
5 Applications
5.1 Diagnosticuses
5.2 Microbiologicaluses
5.3 Usesinresearch
5.4 Detectionofphytopathogens
5.5 Detectionofgeneticallymodifiedorganisms
5.6 Clinicalquantificationandgenotyping
6 References
7 Bibliography
8 Externallinks
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Background
Cellsinallorganismsregulategeneexpressionbyturnoverof
genetranscripts(singlestrandedRNA):Theamountofan
expressedgeneinacellcanbemeasuredbythenumberof
copiesofanRNAtranscriptofthatgenepresentinasample.
Inordertorobustlydetectandquantifygeneexpressionfrom
smallamountsofRNA,amplificationofthegenetranscriptis
necessary.Thepolymerasechainreaction(PCR)isacommon
methodforamplifyingDNAforRNAbasedPCRtheRNA
sampleisfirstreversetranscribedtocomplementaryDNA
(cDNA)withreversetranscriptase.
InordertoamplifysmallamountsofDNA,thesame
methodologyisusedasinconventionalPCRusingaDNA
RealtimePCRusesfluorophoresinordertodetect
template,atleastonepairofspecificprimers,
levelsofgeneexpression.
deoxyribonucleotides,asuitablebuffersolutionandathermo
stableDNApolymerase.Asubstancemarkedwitha
fluorophoreisaddedtothismixtureinathermalcyclerthatcontainssensorsformeasuringthefluorescenceofthe
flurophoreafterithasbeenexcitedattherequiredwavelengthallowingthegenerationratetobemeasuredforone
ormorespecificproducts.ThisallowstherateofgenerationoftheamplifiedproducttobemeasuredateachPCR
cycle.Thedatathusgeneratedcanbeanalysedbycomputersoftwaretocalculaterelativegeneexpression(or
mRNAcopynumber)inseveralsamples.QuantitativePCRcanalsobeappliedtothedetectionandquantification
ofDNAinsamplestodeterminethepresenceandabundanceofaparticularDNAsequenceinthesesamples.[3]
Thismeasurementismadeaftereachamplificationcycle,andthisisthereasonwhythismethodiscalledrealtime
PCR(thatis,immediateorsimultaneousPCR).InthecaseofRNAquantitation,thetemplateiscomplementary
DNA(cDNA),whichisobtainedbyreversetranscriptionofribonucleicacid(RNA).Inthisinstancethetechnique
usedisquantitativeRTPCRorQRTPCR.
QuantitativePCRandDNAmicroarrayaremodernmethodologiesforstudyinggeneexpression.Oldermethods
wereusedtomeasuremRNAabundance:Differentialdisplay,RNaseprotectionassayandNorthernblot.Northern
blottingisoftenusedtoestimatetheexpressionlevelofagenebyvisualizingtheabundanceofitsmRNA
transcriptinasample.Inthismethod,purifiedRNAisseparatedbyagarosegelelectrophoresis,transferredtoa
solidmatrix(suchasanylonmembrane),andprobedwithaspecificDNAorRNAprobethatiscomplementaryto
thegeneofinterest.Althoughthistechniqueisstillusedtoassessgeneexpression,itrequiresrelativelylarge
amountsofRNAandprovidesonlyqualitativeorsemiquantitativeinformationofmRNAlevels.[4]Estimation
errorsarisingfromvariationsinthequantificationmethodcanbetheresultofDNAintegrity,enzymeefficiency
andmanyotherfactors.Forthisreasonanumberofstandardizationsystemshavebeendeveloped.Somehavebeen
developedforquantifyingtotalgeneexpression,butthemostcommonareaimedatquantifyingthespecificgene
beingstudiedinrelationtoanothergenecalledanormalizinggene,whichisselectedforitsalmostconstantlevel
ofexpression.Thesegenesareoftenselectedfromhousekeepinggenesastheirfunctionsrelatedtobasiccellular
survivalnormallyimplieconstitutivegeneexpression.[5][6]Thisenablesresearcherstoreportaratioforthe
expressionofthegenesofinterestdividedbytheexpressionoftheselectednormalizer,therebyallowing
comparisonoftheformerwithoutactuallyknowingitsabsolutelevelofexpression.
Themostcommonlyusednormalizinggenesarethosethatcodeforthefollowingmolecules:tubulin,
glyceraldehyde3phosphatedehydrogenase,albumin,cyclophilin,andribosomalRNAs.[4]

Basicprinciples
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RealtimePCRiscarriedoutinathermalcyclerwiththecapacitytoilluminateeachsamplewithabeamoflight
ofatleastonespecifiedwavelengthanddetectthefluorescenceemittedbytheexcitedfluorophore.Thethermal
cyclerisalsoabletorapidlyheatandchillsamples,therebytakingadvantageofthephysicochemicalpropertiesof
thenucleicacidsandDNApolymerase.
ThePCRprocessgenerallyconsistsofaseriesoftemperaturechangesthatarerepeated2550times.These
cyclesnormallyconsistofthreestages:thefirst,ataround95C,allowstheseparationofthenucleicacidsdouble
chainthesecond,atatemperatureofaround5060C,allowsthebindingoftheprimerswiththeDNA
template[7]thethird,atbetween6872C,facilitatesthepolymerizationcarriedoutbytheDNApolymerase.
DuetothesmallsizeofthefragmentsthelaststepisusuallyomittedinthistypeofPCRastheenzymeisableto
increasetheirnumberduringthechangebetweenthealignmentstageandthedenaturingstage.Inaddition,infour
stepsPCRthefluorescenceismeasuredduringshorttemperaturephaselastingonlyafewsecondsineachcycle,
withatemperatureof,forexample,80C,inordertoreducethesignalcausedbythepresenceofprimerdimers
whenanonspecificdyeisused.[8]Thetemperaturesandthetimingsusedforeachcycledependonawidevariety
ofparameters,suchas:theenzymeusedtosynthesizetheDNA,theconcentrationofdivalentionsand
deoxyribonucleotides(dNTPs)inthereactionandthebondingtemperatureoftheprimers.[9]

Classification
ThetypeofrealtimePCRtechniqueuseddependsontheDNAsequenceinthesamples,thetechniquecaneither
usenonspecificfluorochromesorhybridizationprobes.

RealtimePCRwithdoublestrandedDNAbindingdyesasreporters
ADNAbindingdyebindstoalldoublestranded(ds)DNAinPCR,causingfluorescenceofthedye.Anincrease
inDNAproductduringPCRthereforeleadstoanincreaseinfluorescenceintensitymeasuredateachcycle.
However,dsDNAdyessuchasSYBRGreenwillbindtoalldsDNAPCRproducts,includingnonspecificPCR
products(suchasPrimerdimer).Thiscanpotentiallyinterferewith,orprevent,accuratemonitoringofthe
intendedtargetsequence.
InrealtimePCRwithdsDNAdyesthereactionispreparedasusual,withtheadditionoffluorescentdsDNAdye.
ThenthereactionisruninarealtimePCRinstrument,andaftereachcycle,theintensityoffluorescenceis
measuredwithadetectorthedyeonlyfluoresceswhenboundtothedsDNA(i.e.,thePCRproduct).Thismethod
hastheadvantageofonlyneedingapairofprimerstocarryouttheamplification,whichkeepscostsdown
however,onlyonetargetsequencecanbemonitoredinatube.

Fluorescentreporterprobemethod
FluorescentreporterprobesdetectonlytheDNAcontainingthesequencecomplementarytotheprobetherefore,
useofthereporterprobesignificantlyincreasesspecificity,andenablesperformingthetechniqueeveninthe
presenceofotherdsDNA.Usingdifferentcolouredlabels,fluorescentprobescanbeusedinmultiplexassaysfor
monitoringseveraltargetsequencesinthesametube.Thespecificityoffluorescentreporterprobesalsoprevents
interferenceofmeasurementscausedbyprimerdimers,whichareundesirablepotentialbyproductsinPCR.
However,fluorescentreporterprobesdonotpreventtheinhibitoryeffectoftheprimerdimers,whichmaydepress
accumulationofthedesiredproductsinthereaction.
ThemethodreliesonaDNAbasedprobewithafluorescentreporteratoneendandaquencheroffluorescenceat
theoppositeendoftheprobe.Thecloseproximityofthereportertothequencherpreventsdetectionofits
fluorescencebreakdownoftheprobebythe5'to3'exonucleaseactivityoftheTaqpolymerasebreaksthe
reporterquencherproximityandthusallowsunquenchedemissionoffluorescence,whichcanbedetectedafter
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(1)Inintactprobes,reporterfluorescenceisquenched.(2)ProbesandthecomplementaryDNAstrandarehybridizedandreporter
fluorescenceisstillquenched.(3)DuringPCR,theprobeisdegradedbytheTaqpolymeraseandthefluorescentreporterreleased.

excitationwithalaser.AnincreaseintheproducttargetedbythereporterprobeateachPCRcyclethereforecauses
aproportionalincreaseinfluorescenceduetothebreakdownoftheprobeandreleaseofthereporter.
1.ThePCRispreparedasusual(seePCR),andthereporterprobeisadded.
2.Asthereactioncommences,duringtheannealingstageofthePCRbothprobeandprimersannealtothe
DNAtarget.
3.PolymerisationofanewDNAstrandisinitiatedfromtheprimers,andoncethepolymerasereachesthe
probe,its5'3'exonucleasedegradestheprobe,physicallyseparatingthefluorescentreporterfromthe
quencher,resultinginanincreaseinfluorescence.
4.FluorescenceisdetectedandmeasuredinarealtimePCRmachine,anditsgeometricincrease
correspondingtoexponentialincreaseoftheproductisusedtodeterminethequantificationcycle(Cq)in
eachreaction.

Fusiontemperatureanalysis
RealtimePCRpermitstheidentificationofspecific,amplifiedDNAfragmentsusinganalysisoftheirmelting
temperature(alsocalledTmvalue,frommeltingtemperature).ThemethodusedisusuallyPCRwithdouble
strandedDNAbindingdyesasreportersandthedyeusedisusuallySYBRGreen.TheDNAmeltingtemperature
isspecifictotheamplifiedfragment.Theresultsofthistechniqueareobtainedbycomparingthedissociation
curvesoftheanalysedDNAsamples.[11]
UnlikeconventionalPCR,thismethodavoidstheprevioususeofelectrophoresistechniquestodemonstratethe
resultsofallthesamples.Thisisbecause,despitebeingakinetictechnique,quantitativePCRisusuallyevaluated
atadistinctendpoint.Thetechniquethereforeusuallyprovidesmorerapidresultsand/orusesfewerreactants
thanelectrophoresis.Ifsubsequentelectrophoresisisrequireditisonlynecessarytotestthosesamplesthatreal
timePCRhasshowntobedoubtfuland/ortoratifytheresultsforsamplesthathavetestedpositiveforaspecific
determinant.

Quantificationofgeneexpression
QuantifyinggeneexpressionbytraditionalDNAdetectionmethodsisunreliable.DetectionofmRNAona
NorthernblotorPCRproductsonagelorSouthernblotdoesnotallowprecisequantification.[12]Forexample,
overthe2040cyclesofatypicalPCR,theamountofDNAproductreachesaplateauthatisnotdirectlycorrelated
withtheamountoftargetDNAintheinitialPCR.

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RealtimePCRcanbeusedtoquantifynucleicacidsbytwocommon
methods:relativequantificationandabsolutequantification.[13]Absolute
quantificationgivestheexactnumberoftargetDNAmoleculesby
comparisonwithDNAstandardsusingacalibrationcurve.Itistherefore
essentialthatthePCRofthesampleandthestandardhavethesame
amplificationefficiency.Relativequantificationisbasedoninternal
referencegenestodeterminefolddifferencesinexpressionofthetarget
gene.Thequantificationisexpressedasthechangeinexpressionlevelsof
mRNAinterpretedascomplementaryDNA(cDNA,generatedbyreverse
transcriptionofmRNA).Relativequantificationiseasiertocarryoutasit
doesnotrequireacalibrationcurveastheamountofthestudiedgeneis
comparedtotheamountofacontrolreferencegene.
Astheunitsusedtoexpresstheresultsofrelativequantificationare
unimportanttheresultscanbecomparedacrossanumberofdifferentRT
QPCR.Thereasonforusingoneormorehousekeepinggenesistocorrect
nonspecificvariation,suchasthedifferencesinthequantityandqualityof
RNAused,whichcanaffecttheefficiencyofreversetranscriptionand
thereforethatofthewholePCRprocess.However,themostcrucialaspect
oftheprocessisthatthereferencegenemustbestable.[14]

Distinctfusioncurvesforanumber
ofPCRproducts(showingdistinct
colours).Amplificationreactionscan
beseenforaspecificproduct(pink,
blue)andotherswithanegativeresult
(green,orange).Thefusionpeak
indicatedwithanarrowshowsthe
peakcausedbyprimerdimers,which
isdifferentfromtheexpected

Theselectionofthesereferencegeneswastraditionallycarriedoutin
molecularbiologyusingqualitativeorsemiquantitativestudiessuchasthe
visualexaminationofRNAgels,Northernblotdensitometryorsemi
amplificationproduct. [10]
quantitativePCR(PCRmimics).Now,inthegenomeera,itispossibleto
carryoutamoredetailedestimateformanyorganismsusingDNA
microarrays.[15]However,researchhasshownthatamplificationofthemajorityofreferencegenesusedin
quantifyingtheexpressionofmRNAvariesaccordingtoexperimentalconditions.[16][17][18]Itistherefore
necessarytocarryoutaninitialstatisticallysoundmethodologicalstudyinordertoselectthemostsuitable
referencegene.

Anumberofstatisticalalgorithmshavebeendevelopedthatcandetectwhichgeneorgenesaremostsuitablefor
useundergivenconditions.ThoselikegeNORMorBestKeepercancomparepairsorgeometricmeansfora
matrixofdifferentreferencegenesandtissues.[19][20]

Modeling
UnlikeendpointPCR(conventionalPCR)realtimePCRallowsmonitoringofthedesiredproductatanypointin
theamplificationprocessbymeasuringfluorescence(inreality,measurementismadeofitsleveloveragiven
threshold).AcommonlyemployedmethodofDNAquantificationbyrealtimePCRreliesonplottingfluorescence
againstthenumberofcyclesonalogarithmicscale.AthresholdfordetectionofDNAbasedfluorescenceisset3
5timesofthestandarddeviationofthesignalnoiseabovebackground.Thenumberofcyclesatwhichthe
fluorescenceexceedsthethresholdiscalledthethresholdcycle(Ct)or,accordingtotheMIQEguidelines,
quantificationcycle(Cq).[21]
Duringtheexponentialamplificationphase,thequantityofthetargetDNAtemplate(amplicon)doublesevery
cycle.Forexample,aDNAsamplewhoseCqprecedesthatofanothersampleby3cyclescontained23=8times
moretemplate.However,theefficiencyofamplificationisoftenvariableamongprimersandtemplates.Therefore,
theefficiencyofaprimertemplatecombinationisassessedinatitrationexperimentwithserialdilutionsofDNA
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templatetocreateastandardcurveofthechangein(Cq)witheachdilution.Theslopeofthelinearregressionis
thenusedtodeterminetheefficiencyofamplification,whichis100%ifadilutionof1:2resultsina(Cq)
differenceof1.Thecyclethresholdmethodmakesseveralassumptionsofreactionmechanismandhasareliance
ondatafromlowsignaltonoiseregionsoftheamplificationprofilethatcanintroducesubstantialvarianceduring
thedataanalysis.[22]
Toquantifygeneexpression,the(Cq)foranRNAorDNAfromthegeneofinterestissubtractedfromthe(Cq)of
RNA/DNAfromahousekeepinggeneinthesamesampletonormalizeforvariationintheamountandqualityof
RNAbetweendifferentsamples.ThisnormalizationprocedureiscommonlycalledtheCtmethod[23]andpermits
comparisonofexpressionofageneofinterestamongdifferentsamples.However,forsuchcomparison,expression
ofthenormalizingreferencegeneneedstobeverysimilaracrossallthesamples.Choosingareferencegene
fulfillingthiscriterionisthereforeofhighimportance,andoftenchallenging,becauseonlyveryfewgenesshow
equallevelsofexpressionacrossarangeofdifferentconditionsortissues.[24][25]Althoughcyclethresholdanalysis
isintegratedwithmanycommercialsoftwaresystems,therearemoreaccurateandreliablemethodsofanalysing
amplificationprofiledatathatshouldbeconsideredincaseswherereproducibilityisaconcern.[22]
MechanismbasedqPCRquantificationmethodshavealsobeensuggested,andhavetheadvantagethattheydonot
requireastandardcurveforquantification.MethodssuchasMAK2[26]havebeenshowntohaveequalorbetter
quantitativeperformancetostandardcurvemethods.Thesemechanismbasedmethodsuseknowledgeaboutthe
polymeraseamplificationprocesstogenerateestimatesoftheoriginalsampleconcentration.Anextensionofthis
approachincludesanaccuratemodeloftheentirePCRreactionprofile,whichallowsfortheuseofhighsignalto
noisedataandtheabilitytovalidatedataqualitypriortoanalysis.[22]
AccordingtoresearchofRuijteretal.[27]MAK2assumesconstantamplificationefficiencyduringthePCR
reaction.However,theoreticalanalysisofpolymerasechainreaction,fromwhichMAK2wasderived,hasrevealed
thatamplificationefficiencyisnotconstantthroughoutPCR.WhileMAK2quantificationprovidesreliable
estimatesoftargetDNAconcentrationinasampleundernormalqPCRconditions,MAK2doesnotreliably
quantifytargetconcentrationforqPCRassayswithcompetimeters.

Applications
Therearenumerousapplicationsforquantitativepolymerasechainreactioninthelaboratory.Itiscommonlyused
forbothdiagnosticandbasicresearch.Usesofthetechniqueinindustryincludethequantificationofmicrobial
loadinfoodsoronvegetablematter,thedetectionofGMOs(Geneticallymodifiedorganisms)andthe
quantificationandgenotypingofhumanviralpathogens.

Diagnosticuses
DiagnosticqualitativePCRisappliedtorapidlydetectnucleicacidsthatarediagnosticof,forexample,infectious
diseases,cancerandgeneticabnormalities.TheintroductionofqualitativePCRassaystotheclinicalmicrobiology
laboratoryhassignificantlyimprovedthediagnosisofinfectiousdiseases,[28]andisdeployedasatooltodetect
newlyemergingdiseases,suchasnewstrainsofflu,indiagnostictests.[29]

Microbiologicaluses

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QuantitativePCRisalsousedbymicrobiologistsworkinginthefieldsoffoodsafety,foodspoilageand
fermentationandforthemicrobialriskassessmentofwaterquality(drinkingandrecreationalwaters)andinpublic
healthprotection.[30]

Usesinresearch
Inresearchsettings,quantitativePCRismainlyusedtoprovidequantitativemeasurementsofgenetranscription.
Thetechnologymaybeusedindetermininghowthegeneticexpressionofaparticulargenechangesovertime,
suchasintheresponseoftissueandcellculturestoanadministrationofapharmacologicalagent,progressionof
celldifferentiation,orinresponsetochangesinenvironmentalconditions.Itisalsousedforthedeterminationof
zygosityoftransgenicanimalsusedinresearch.

Detectionofphytopathogens
Theagriculturalindustryisconstantlystrivingtoproduceplantpropagulesorseedlingsthatarefreeofpathogens
inordertopreventeconomiclossesandsafeguardhealth.Systemshavebeendevelopedthatallowdetectionof
smallamountsoftheDNAofPhytophthoraramorum,anoomycetethatkillsOaksandotherspecies,mixedin
withtheDNAofthehostplant.DiscriminationbetweentheDNAofthepathogenandtheplantisbasedonthe
amplificationofITSsequences,spacerslocatedinribosomalRNAgenescodingarea,whicharecharacteristicfor
eachtaxon.[31]Fieldbasedversionsofthistechniquehavealsobeendevelopedforidentifyingthesame
pathogen.[32]

Detectionofgeneticallymodifiedorganisms
qPCRusingreversetranscription(RTqPCR)canbeusedtodetectGMOsgivenitssensitivityanddynamicrange
indetectingDNA.AlternativessuchasDNAorproteinanalysisareusuallylesssensitive.Specificprimersare
usedthatamplifynotthetransgenebutthepromoter,terminatororevenintermediatesequencesusedduringthe
processofengineeringthevector.Astheprocessofcreatingatransgenicplantnormallyleadstotheinsertionof
morethanonecopyofthetransgeneitsquantityisalsocommonlyassessed.Thisisoftencarriedoutbyrelative
quantificationusingacontrolgenefromthetreatedspeciesthatisonlypresentasasinglecopy.[33][34]

Clinicalquantificationandgenotyping
Virusescanbepresentinhumansduetodirectinfectionorcoinfectionswhichmakesdiagnosisdifficultusing
classicaltechniquesandcanresultinanincorrectprognosisandtreatment.TheuseofqPCRallowsboththe
quantificationandgenotyping(characterizationofthestrain,carriedoutusingmeltingcurves)ofavirussuchas
theHepatitisBvirus.[35]Thedegreeofinfection,quantifiedasthecopiesoftheviralgenomeperunitofthe
patientstissue,isrelevantinmanycasesforexample,theprobabilitythatthetype1herpessimplexvirus
reactivatesisrelatedtothenumberofinfectedneuronsintheganglia.[36]Thisquantificationiscarriedouteither
withreversetranscriptionorwithoutit,asoccursifthevirusbecomesintegratedinthehumangenomeatanypoint
initscycle,suchashappensinthecaseofHPV(humanpapillomavirus),wheresomeofitsvariantsareassociated
withtheappearanceofcervicalcancer.[37]

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https://en.wikipedia.org/wiki/Realtime_polymerase_chain_reaction

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RealtimepolymerasechainreactionWikipedia,thefreeencyclopedia

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9781908230010(http://www.horizonpress.com/qpcr)
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Externallinks
BeginnersGuidetoRealTimePCRbyPrimerdesign(http://www.primerdesign.co.uk/assets/files/beginners_
guide_to_real_time_pcr.pdf)
TheReferenceinqPCRanAcademic&IndustrialInformationPlatform(http://www.genequantification.i
nfo/)
www.eConferences.deAmplifyyourknowledgeinqPCR,dPCRandNGS!(http://www.eConferences.de/)
RealTimePCRTutorial(http://pathmicro.med.sc.edu/pcr/realtimehome.htm)byDrMargaretHunt,
UniversityofSouthCarolina,September5,2006
openwetware(http://openwetware.org/wiki/QPCR)
OnlinePCR.com(http://www.onlinepcr.com)Openonlinetoolforqualitativeandquantitativeanalysisof
realtimePCRdata.UsesbothstandardcurveandMAK2method.
RefGenes(http://www.refgenes.org)OpenAccessonlinetooltoidentifytissuespecificreferencegenesfor
RTqPCR.
RealtimePCRuserexperiences(http://www.realtimepcr.dk)
ArticlesaboutRealTimePcr(http://polymerasechainreaction.org/category/realtimepcr/)
BasicsaboutqPCR(http://biosistemika.com/workshops/qpcrbasics/)
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