Selection of Suitable Reference Genes for qPCR Normalization under Abiotic Stresses and ...

16 Pages·2015·2.26 MB·English

RESEARCHARTICLE
Selection of Suitable Reference Genes for
qPCR Normalization under Abiotic Stresses
and Hormone Stimuli in Carrot Leaves
ChangTian,QianJiang,FengWang,Guang-LongWang,Zhi-ShengXu,Ai-ShengXiong*
StateKeyLaboratoryofCropGeneticsandGermplasmEnhancement,CollegeofHorticulture,Nanjing
AgriculturalUniversity,Nanjing,210095,China
* [email protected]
Abstract
Carrot,abiennialherboftheApiaceaefamily,isamongthemostimportantvegetablecrops
intheworld.Inthisstudy,ninecandidatereferencegenes(GAPDH,ACTIN,eIF-4α,PP2A,
SAND,TIP41,UBQ,EF-1α,andTUB)wereclonedfromcarrot.Carrotplantsweresubjected
OPENACCESS toabioticstresses(heat,cold,salt,anddrought)andhormonestimuli(gibberellin,salicylic
Citation:TianC,JiangQ,WangF,WangG-L,XuZ- acid,methyljasmonate,andabscisicacid).Theexpressionprofilesofthecandidaterefer-
S,XiongA-S(2015)SelectionofSuitableReference encegeneswereevaluatedinthreetechnicalandbiologicalreplicates.Real-timeqPCR
GenesforqPCRNormalizationunderAbiotic
dataanalyseswereperformedusingthreecommonlyusedExcel-basedappletsnamely,
StressesandHormoneStimuliinCarrotLeaves.
BestKeeper,geNorm,andNormFinder.ACTINandTUBwerethemoststablegenesidenti-
PLoSONE10(2):e0117569.doi:10.1371/journal.
pone.0117569 fiedamongallsamplegroups,butindividualanalysisrevealedchangesintheirexpression
profiles.GAPDHdisplayedthemaximumstabilityformostofsinglestresses.Tofurthervali-
AcademicEditor:MukeshJain,NationalInstituteof
PlantGenomeResearch,INDIA datethesuitabilityofthereferencegenesidentifiedinthisstudy,theexpressionprofileof
DcDREB-A1gene(homologofAtDREB-A1geneofArabidophsis)wasstudiedincarrot.
Received:September16,2014
Theappropriatereferencegeneswereselectedthatshowedstableexpressionunderthe
Accepted:December28,2014
differentexperimentalconditions.
Published:February6,2015
Copyright:©2015Tianetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
Introduction
credited.
DataAvailabilityStatement:Allrelevantdataare Quantitativereal-timereversetranscriptionpolymerasechainreaction(qPCR)allowsaccurate
withinthepaperanditsSupportingInformationfiles. high-throughputRNAquantificationoverawidedynamicrangeatarelativelylowcost;this
techniquehashighsensitivityandhasbeenwidelyusedforgeneexpressionanalysis[1–4].Ap-
Funding:TheresearchwassupportedbytheNew
CenturyExcellentTalentsinUniversity(NCET-11- propriatereferencegenescouldeliminatethediscrepancythatmayexistindifferentsamples
0670);JiangsuNaturalScienceFoundation andensuretheaccuracyandreliabilityoftheexperimentalresults.Discrepanciesmaybedue
(BK20130027);ChinaPostdoctoralScience tovariationsinRNAexpressionlevelsandthequalityandefficiencyofreversetranscription.
Foundation(2014M551609);PriorityAcademic
Theuseofreferencegenestomeasurethetemporalandspatialexpressionsofthetargetgeneis
ProgramDevelopmentofJiangsuHigherEducation
widelyacknowledgedasastandardizedmethod.Inhigherplants,suitableinternalcontrolsfor
Institutions.Thefundershadnoroleinstudydesign,
geneexpressionstudieshavebeenrecognizedforpepper[5],rice[6],Arabidopsisthaliana[7],
datacollectionandanalysis,decisiontopublish,or
preparationofthemanuscript. Brachypodiumdistachyon[8],chicory[9],poplar[10],coffee[11],Oenanthejavanica(BI.)
PLOSONE|DOI:10.1371/journal.pone.0117569 February6,2015 1/16
ReferenceGenesinCarrotLeaves
CompetingInterests:Theauthorshavedeclared [12],andpeach[13].Asofthiswriting,nosystematicstrategyisavailabletoanalyzecarrotref-
thatnocompetinginterestsexist. erencegenesunderabioticstressandhormonestimuliconditions.
Hundredsofpotentialhousekeepinggeneshavebeenidentifiedbymicroarrayanalysesin
Arabidopsis[7].However,previousstudiesindicatedthatgenescommonlyusedasinternal
controlsare3-glyceraldehydephosphatedehydrogenase(GAPDH),translationelongationfac-
torEF-1alpha(EF-1α),poly-ubiquitin(UBQ),actin(ACTIN),andtubulin(TUB)[14–20].
Thesegenesreferredtoashousekeepingcontrolgenesandplayedhousekeepingrolesinbasic
cellularprocesses,suchascellstructuremaintenanceorprimarymetabolism[7],althoughwe
referthemheresimplyasreferencegenes.Currently,somenewreferencegenesarewellde-
scribedforthenormalizationofexpressionsignalsincludingproteinphosphatase2A(PP2A),
genesencodingF-box/kelch-repeatprotein(F-box),SANDfamilyprotein(SAND),Eukaryotic
translationinitiationfactor4α(eIF-4α),andTap42-inter-actingproteinof41kDa(TIP41)
[7,21–23].However,severalstudieshavescrutinizedthatsomecommonlyusedreference
geneslikeACTINandGAPDHshoweddifferentbehaviorsindifferentplants,tissues,andex-
perimentconditions,andtheseshouldbeusedwithcautionasinternalcontrols[24,25].The
reasonfortheseexpressionalvariabilitiesmaybethattranscriptlevelsofreferencegenescould
varyconsiderablyinresponsetoexperimentalconditions,cellularprocess,andtissuetypes
[26–28].Thenormalizationwillproducemisleadingresults,iftheselectedreferencegenehasa
largeexpressionfluctuation[13].Hence,theappropriatereferencegenesforqPCRmustbese-
lectedtoobtainnormalizationofRNAquantitationandexperimentaldataindifferentsamples
andtoensuretheaccuracyandreliabilityoftheexperimentalresults.Moreover,theoptimal
numberofreferencegenesshouldbedeterminedandmultiplereferencegenesarerequiredfor
geneexpressionstudyinsteadofasinglegene[29].
Carrot(DaucuscarotaL.)isabiennialherbcultivatedaroundtheworldandbelongsto
theDaucusgenusintheApiaceaefamily(Fig.AandBinS1File).Carrotcontainsabundant
β-carotenewhichimportsbenificialpropertiestohumanhealth,likeanti-cancer,antioxidant,
detoxification,cardiovascularprotection,cataractpreventionandtreatment,andliverpro-
tection[30–33].Phytohormonesincludingsalicylicacid(SA),methyljasmonate(MeJA),
gibberellicacid(GA),andabscisicacid(ABA),areknowntoplayimportantrolesintheregula-
tionofplantdevelopmentalprocesses,andresponsestobioticandabioticstresses[34,35].Ex-
ogenousSAcouldincreaseplanttolerancetotheabioticstressbyregulatingtheactivitiesof
antioxidantenzymes[36].Incarrot,SAhasbeenshowntopositivelyaffectthecarotenoids
andanthocyanincontent,storagerootdryweight,andincreasethetotalantioxidantactivity
oftheshootandstorageroot[37].MeJAtreatmentcouldincreasethecontentofphytoalexin
6-methoxymellin[38].ExogenousGAcouldbeappliedinvernalizationtopreventtheinhibi-
toryeffectofhightemperatureonseedstalkelongation[39].Moreover,accumulationofABA
couldsuppressesprecociousgerminationandmodulatesseedgeneexpressionindeveloping
seeds[40].Environmentalstressessuchasdrought,highsalt,andtemperaturechangecould
reduceproductivityandsignificantcroplosses,likedroughtandsalinity,whichtogetherresult
inamorethan50%declineintheaverageyieldsofmajorcropsworldwide[41,42].Abiotic
stresses,includingheat,cold,drought,andsalinitytolerance,arealsoknowntolimitcarrot
production[30].
Inthisstudy,ninecandidatereferencegenes(TIP41,TUB,eIF-4α,UBQ,SAND,GAPDH,
EF-1α,PP2A,andACTIN)wereselectedbasedontheirstableexpressioninpreviousstudies
[12,21,28,43,44].Theninegenesequencesofcarrotwereobtainedbasedonthecarrotgenome
sequencedata,whichwasbuiltbyourgroup(LabofApiaceaePlantGeneticsandGermplasm
Enhancement,NanjingAgriculturalUniversity)(http://apiaceae.njau.edu.cn/carrot/).Infor-
mationonthesereferencegenesispresentedinTable1.Threedifferentalgorithms(geNorm,
NormFinder,andBestKeeper)wereusedtoevaluatetheexpressionstabilityofthereference
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ReferenceGenesinCarrotLeaves
Table1.Descriptionsofreferencegenesincarrot(ThelistsofprimersusedinqPCR).
Gene Genename Arabidopsis Primersequence(5’–3’)forward/reverse Amplicon E(%) Tm
symbol homologgene length(bp) L/R (°C)
eIF-4α Eukaryotictranslation AT3G13920 TGTGCTTATCACCACTGACCTTCTG/ 122 108.8 82.5
initiationfactor4α-1gene GTCCACTACGCCCAATACGATGAA
ACTIN Actin1gene AT2G37620 CGGTATTGTGTTGGACTCTGGTGAT/ 98 106.2 82.5
CAGCAAGGTCAAGACGGAGTATGG
TIP41 Tap42-interactingproteinof AT4G34270 GGAGGACTGTGAGGAACGAATTGAT/ 166 101.1 81.0
41kDagene ACGCAAGAGAAGGAACCAACAACT
GAPDH Glyceraldehyde-3- AT1G42970 AGGCTGCTGAAGGACCATTGAAG/ 164 101.2 83.5
phosphatedehydrogenase CCATTCGTTATCGTACCAGGCTACA
gene
SAND SANDfamilyproteingene AT2G28390 AATGCTGCTCACTGCTAATCCAGAT/ 124 96.8 81.0
GCCACCATCCAACATCGACCTC
EF-1α Elongationfactor-1αgene AT1G07940 TCAAGGATCTCAAGCGTGGTTATGT/ 175 100.4 84.0
CAGCAATGTGGCAAGTGTGACAAT
PP2A Proteinphosphatase2A AT4G15415 GTGTATCAATGTACCACCAGCAACT/ 147 97.3 80.0
gene GCTCACCAAGGAACATGACTTCTT
TUB Tubulinbeta-7gene AT2G29550 GAGTGGAGTTACCTGCTGCCTTC/ 94 105.5 84.0
ATGTAGACGAGGGAACGGAATCAAG
UBQ Polyubiquitin10gene AT4G05320 TCTCCGACTCCGTGGTGGTATG/ 180 93.8 85.0
CTGCCGTCCTCCAACTGCTTAC
doi:10.1371/journal.pone.0117569.t001
genes.TheexperimentaldataofthegenesweredeterminedbyqPCRincarrotleavesunderdif-
ferenthormonestimulitreatments(GA,SA,ABA,andMeJA,respectively)andabioticstresses
treatments(heat,cold,salt,anddrought).Allninereferencegenesdisplayedawiderangeof
quantificationcycle(Cq)valuesacrossexperimentalsamples,indicatingvariableexpression.
Furthermore,theexpressionlevelofDcDREB-A1,thehomologofAtDREB-A1(DREB,Dehy-
drationresponsiveelementbindingfactor)geneofArabidopsis,wasassessedusingdifferent
referencegenestovalidatetheselectionofcandidatereferencegenes.Weassumedthattheref-
erencegenesidentifiedincurrentstudywouldenablebetternormalizationandquantification
oftranscriptlevelsinfutureexpressionstudiesoncarrotplants.
MaterialsandMethods
Plantmaterialsandtreatments
SeedsofD.carotavarietyofKurodagosunweresowninplasticpotscontainingasoil/vermicu-
litemixture(1:1)[45–47]andgrowninanartificialclimatechamberprogrammedfor16h/8h
at25°C/16°Cforday/nightconditionsatalightintensityof~300μmol∙m-2∙s-1andrelativehu-
midity60%.Healthyandvigorouseight-week-oldseedlingswereusedfortreatments.In
droughtexperiment,soilwereirrigatedwith500mLof20%PEG6000for2hineachpots.In
saltexperiment,leavesweresprayedwith500mLof0.2MNaClfor2h.Coldandheattreat-
mentswereperformedbyexposingeight-week-oldseedlingsto4and40°Ctemperaturesin
lightincubatorsfor2h,respectively.Forhormonetreatments,leavesweresprayedwith500
mLofSA(1.4mM)[37,48],MeJA(0.8mM)[38],GA(1.4mM)[39],andABA(0.1mM)[40]
for2h,respectively.Plantsweresprayedorirrigatedonlyonce.GA,SA,MeJA(containing
0.02%(v/v)absoluteethanoland0.02%(v/v)Tween-20),andABAweredissolvedindistilled
water[49–53].ThepHofGA,SA,MeJA,andABAdilutionswere2.8,2.8,6.7,and5.3,respec-
tively.Inallcases,potswereplacedinlightincubatorsunderoptimalconditionswithconstant
lightintensity,beingprocessedatthesametimeasplantssubjectedtothedifferentstress
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ReferenceGenesinCarrotLeaves
conditions.Threebiologicalexperimentalreplicateswerecollectedfromthreeseedlingsamples
performedindifferentpotsforeachtreatment.Leaveswerecollectedfromtheeight-week-old
seedlingssubjectedtoalltreatments.Thesampleswerefrozeninliquidnitrogenandstored
at−80°Cuntilfurtheruse.
TotalRNAextractionandcDNAsynthesis
Frozencarrottissuesweredisruptedunderliquidnitrogenconditionsusingmortarandpestle.
TotalRNAextractionwasperformedaccordingtothemanufacturer’sprotocol(Tiangen,Bei-
jing,China).TheconcentrationandpurityofRNAsamplesweremeasuredbyNanoDrop
ND1000spectrophotometer,andcDNAsynthesiswasperformedusinganA /A ratioof
260 280
1.8to2.0samples.Thegeneticintegritywasevaluatedby1.5%agarosegelelectrophoresis.
cDNAwassynthesizedfromapproximately1,000ngtotalRNAusingthePrimeScriptRTre-
agentKitwithgDNAEraser(TaKaRa,Dalian,China).ThecDNAwasten-folddilutedseries
(10×,102×,103×,104×,105×,and106×dilutions)fordeterminingtheamplificationefficiency
(E)andcorrelationcoefficient(R2)analysis;andeighteen-folddilutedforconductingthe
qPCRanalysisofelicitortreatments.
Selectionofcandidatereferencegenesandprimerdesign
Ninegenes,TIP41,TUB,eIF-4α,UBQ,SAND,GAPDH,EF-1α,PP2A,andACTIN,wereused
toidentifythemoststablereferencegenesforqPCRexpressionanalysesoftargetcarrotgenes.
Thesegeneshavealreadybeenidentifiedandhavebeencommonlyusedasinternalcontrolsin
previousstudies[12,21,28,44].Forthisstudy,theArabidopsisgeneswereselectedfromthe
TAIRdatabase(http://www.arabidopsis.org).Potentialhomologsoftheninereferencegenes
wereidentifiedfromthegenomeandtranscriptomedatasequencesofcarrot,whichwerese-
quencedandanalysizedbyourgroup(CarrotDB:http://apiaceae.njau.edu.cn/carrot/)[54].
ThepotentialhomologssequenceswerealignedandeditedbyusingBioEditSequenceAlign-
mentv7.0.9software.PrimersweredesignedusingPrimer6.0(PremierBiosoftInternational,
PaloAlto,CA)andDNAMAN6.0(LynnonBiosoft,USA)accordingtothemanufacturer’sin-
structions.TheprimersusedinqPCR,aswellastheirmeltingtemperatures(80°Cto85°C),
primerlengths(22bpto25bp),GCcontent(44%to60%),andampliconlengths(80bpto
180bp)areprovidedinTable1.CloninginformationispresentedinTableAinS1File.The
specificityoftheampliconswasverifiedbyusingasinglebandofexpectedsizein1.5%agarose
gelfollowingelectrophoresisandbythepresenceofasinglepeakintheqPCRmeltingcurve.
ThetargetampliconsweresequencedtoconfirmspecificityofthePCRproducts.
Quantitativereal-timePCRassay
TheqPCRwasdesignedaccordingtotheminimuminformationforpublicationofquantitative
real-timePCRexperimentguidelines[55].ReactionsusedSYBRGreenIMix(TaKaRa,Dalian,
China)ina20μLreactionvolumeandwereperformedina96-wellplateonMyiQsinglecolor
real-timePCRdetectionsystem(Bio-Rad,Hercules,USA).Reactionmixturescontained10μL
SYBRGreenIMix,2μLdilutedcDNA,ddH O,andafinalprimerconcentrationof0.4μM.
2
Thefollowingamplificationconditionswereapplied:aninitialdenaturationstepof95°Cfor
30s;40cyclesat95°Cfor5s;and60°Cfor20s.Thefinaldissociationcurvewasobtainedfrom
65°Cto95°Ctoverifyprimerspecificity.Eachassayincludedthreetechnicalandbiological
replicates,andastandardcurveofsixserialdilutionpoints.Thegeneralqualityassessmentof
thePCRresultswasbasedontheamplificationandmeltingcurveprofilesofthesamplesinre-
lationtotheassaycontrols(non-templatecontrols).MeanCqvaluesoftheten-folddilutionse-
rieswereplottedagainstthelogarithmofthepooledcDNAdilutionfactors.TheCqvaluesand
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thefollowingequationwereusedtodetermineefficiency(E)ofeachgenewiththeslopeofa
linearregressionmodel:%E=(10[−1/slope]-1)×100%[56].Amplificationefficiencieswerecal-
culatedfromstandardcurveswithsatisfactorylinearrelationships(R2>0.99).AllPCRpro-
cessesdisplayedefficiencybetween90%and110%.
Dataanalysis
ThreedifferenttypesofMicrosoftExcel-basedsoftware,namely,geNorm[29],NormFinder
[57],andBestKeeper[58],wereusedtoranktheexpressionstabilityofreferencegenesacross
allexperimentalsets.Thesedatawereeitheruseddirectlyforstabilitycalculations(BestKeeper
analysis)orwereconvertedintorelativequantitiesandimportedintothegeNormandNorm-
Finderusingtheformula2-ΔCq,inwhichΔCq=thecorrespondingCqvalue—minimumCq.
TherawdataarelistedinTableBinS1File.
IngeNorm,thereferencegeneexpressionstabilitymeasurement(M)valueiscalculatedas
thelevelofpairwisevariationforeachreferencegenewithallothercontrolgenesandasthe
standarddeviation(SD)ofthelogarithmicallytransformedexpressionratios[29].Therefer-
encegenewiththelowestMvalueisconsideredthemoststablegene[59].SimilartogeNorm,
theNormFinderprogramisanotherVisualBasicapplicationtoolforMicrosoftExcelthatis
usedtodeterminetheexpressionstabilitiesofreferencegenes[12].Misinterpretationscaused
byartificialselectionofco-regulatedgenesareavoidedwiththisprogram[57].BestKeeperde-
terminesthemoststablyexpressedgenesbasedonthecoefficientofcorrelationtothecandi-
datereferencegene’sCqvalues[58].GeneswiththelowestSDandCVvaluesarethemost
stable[60].
Results
Cqvaluesofcandidatereferencegenesincarrot
BasedonprimersequencesfromTableAinS1File,cDNAofninegeneswereclonedandiden-
tifiedincarrotleavesbasedonthedataofcarrotgenomeandtranscriptomesequences.The
geneexpressionlevelsweredeterminedasCqvalues(TableBinS1File),andthetranscriptsof
thereferencegenesshoweddifferentlevelsofabundance(Fig.1).MeanCqvaluesofthegenes
rangedfrom24.49(EF-1α)to32.96(TIP41),andtheCqvaluesofallthetestedsampleswere
between18.62(EF-1α)and38.01(TIP41).LowCqvaluescorrespondedtohighlevelsofex-
pression.EF-1αshowedhighexpressionlevelwithlowCqvalue.TIP41andSANDshowedlow
expressionlevelswithhighCqvalues(Fig.2).
Determinationoftheoptimalnumberofreferencegenesincarrot
TheoptimalnumberofreferencegenesrequiredfornormalizationwasdeterminedwithgeN-
ormusingpairwisevariations(Vn/n+1)betweenthesequentialnormalizationfactors(NFn
andNFn+1,n(cid:1)2).Alargevariationbetweenthesequentialnormalizationfactorsindicates
thattheaddedgenehasasignificanteffectandispreferredforinclusionandcalculationofare-
liablenormalizationfactor[29].AsshowninFig.3,thethirdgenehadnosignificanteffect
(V ,lowvalue)incoldanddroughtconditions.Thus,tworeferencegenesweresufficientfor
2/3
normalizinggeneexpressionunderthecoldanddroughtconditions.Withathresholdof0.15,
threegenesweresufficientfornormalizinggeneexpressionunderheatandGAstresscondi-
tions,fiveforSAstressandsixforMeJAstress.Noneofthegeneselectedwasfoundtobeap-
propriateinsaltstressconditioninthecurrentstudy.
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ReferenceGenesinCarrotLeaves
Fig1.Cqvaluesofcandidatereferencegenesinallcarrotsamples.Asterisksdenoteoutliers.Thelineacrosstheboxdepictsthemedianvalue.The
insideboxdepictsCqvalues.Theoutsidebox’sbottomlineisdeterminedbythe25thpercentile,whereasthetoplineisdeterminedbythe75thpercentile.
Thetopandbottomwhiskersaredeterminedbythe5thand95thpercentiles,respectively.
doi:10.1371/journal.pone.0117569.g001
Expressionstabilityofcandidatereferencegenesincarrot
Threedifferentsoftwareprogramswereusedtocalculatetheexpressionstabilityofthecandi-
datereferencegenes:geNorm,NormFinder,andBestKeeper.Eightdifferenttreatmentsets
weresortedintothreegroups:“abioticstress”(heat,cold,salt,anddrought),“hormonestimuli”
(SA,GA,ABA,andMeJA),and“total”(samplesinalltreatments).Accordingly,11evaluation
patternsweregeneratedforbothsinglestresstreatmentsandgroups.
AccordingtogeNorm,inwhichthedefaultlimitcomprisedMvalueslessthan1.5,andex-
ceptforTIP41underABAstress,alltheotherreferencegenesperformedwellunderindividual
stressconditions(TableCinS1File).EF-1αandACTINwerethetwobestgenesamongthe
ninereferencegenesinSAandsaltstresstreatments.However,intheMeJAtreatment,EF-1α
andUBQwerethetwobestreferencegenes.InNormFinder,TIP41wasthemoststablegene
amongtheninecandidategenesundersaltandSAstressconditions.UBQwasthemoststable
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ReferenceGenesinCarrotLeaves
Fig2.DatastatisticsofCqvaluesofcandidatereferencegenesincarrot.TotalnumberofCqvaluesineachreferencegenesis72.Mean,median,
minimum,andmaximunofCqvaluesweredeterminedbystatisticanalysis.SDoftheCqvaluesweregeneratedbyBestKeeper.
doi:10.1371/journal.pone.0117569.g002
geneunderGAandcoldstressconditions.TheBestKeeperanalysisshowedthatmostofthe
ninecandidategeneshadsatisfactorystability.TUB,GAPDH,andUBQwererankedatthetop
positionsinmostofthesinglestresstreatmentsbyBestKeeperanalysis.Allcandidategenes
wereconfirmedtobestableunderGAtreatmentinBestKeeper.
Recognizingthebestreferencegenewasdifficultbecauseofthecomplexityofthegroups.
TheresultsoftheanalysisofthethreegroupsofsamplesareshowninTable2.Theninecandi-
dategenesperformedwellbygeNormanalysis.Inthe“abioticstress”group,ACTINandUBQ
werethetwomoststablegenes,andeIF-4αandGAPDHrankedtoptwointhe“hormonesti-
muli”group.ACTINandEF-1αwerethetwomoststablegenesinthe“total”group.ACTIN,
EF-1α,andGAPDHperformedwellinallthreegroupsbygeNormanalysis.eIF-4αwasthe
moststablereferencegenewiththeminimumvalueof0.005obtainedbyNormFinderinthe
“hormonestimuli”group.ACTINwasthemoststablereferencegenewiththevalueof0.012
and0.015obtainedbyNormFinderin“total”and“abioticstress”groups,whereasitranked
fourthin“hormonestimuli”group.GAPDHperformedwellin“hormonestimuli”groupby
NormFinderanalysis,whileitrankedthelastonein“abioticstress”and“total”group.Inall
threegroups,ACTIN,eIF-4α,andTIP41performedwellintermsofstabilityaccordingto
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ReferenceGenesinCarrotLeaves
Fig3.Determinationoftheoptimalnumberofreferencegenes.Pairwisevariation(V )analysisbetweenthenormalizationfactors(NF andNF )
n/n+1 n n+1
wasperformedbyusingthegeNormprograminallsamplesMeJA,methyljasmonate;SA,salicylicacid;GA,gibberellin;andABA,abscisicacid.Theabiotic
stressgroupincludedheat,cold,drought,andsalttreatments.ThehormonestimuligroupincludeSA,GA,ABA,andMeJA.Thetotalgroupincluded
allsamples.
doi:10.1371/journal.pone.0117569.g003
NormFinderanalysis.InBestKeeper,EF-1αwasthemoststablereferencegenein“abiotic
stress”group,whereasitwastheleaststablein“hormonestimuli”and“total”groups;PP2A
rankedfirstin“hormonestimuli”and“total”groupanditrankedfifthin“abioticstress”
group.TUBandGAPDHweremorestablethantheothergenesinallthreegroupsaccording
toBestKeeper.Inboth“abioticstress”and“total”groups,ACTINwasrankedfirstaccordingto
geNormandNormFinder;whereasACTINwasrankedsixthandeighthby
BestKeeper,respectively.
Referencegenevalidation
Tovalidatetheselectionofcandidatereferencegenes,therelativeexpressionofDcDREB-A1
wascalculatedbyusingtheselectedreferencegenes(Fig.4).InA.thaliana,theexpressionof
theAtDREB-A1genewasinducedbyabioticstresstreatments,includingcold,drought,and
heat[61–63].Inthisstudy,theexpressionprofilesofDcDREB-A1incarrotunderheatstress
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Table2.Geneexpressionstabilityincarrotundermultiplestresstreatments,asrankedbythethreesoftwareprogramsgeNorm,NormFinder,
andBestKeeper.
Group Rank geNorm NormFinder BestKeeper
Gene Stability Gene Stability Gene SD CV
Abioticstress 1 ACTIN 0.68 ACTIN 0.015 EF-1α 1.06 4.63
2 UBQ 0.68 TIP41 0.015 GAPDH 1.33 5.16
3 EF-1α 0.76 PP2A 0.029 TUB 1.33 4.41
4 GAPDH 0.82 eIF-4α 0.029 UBQ 1.33 4.84
5 PP2A 0.96 SAND 0.039 PP2A 1.36 4.38
6 TUB 1.06 TUB 0.041 ACTIN 1.37 5.32
7 SAND 1.18 EF-1α 0.058 SAND 1.45 4.59
8 eIF-4α 1.28 UBQ 0.060 TIP41 2.00 6.19
9 TIP41 1.36 GAPDH 0.068 eIF-4α 2.25 7.84
Hormonestimuli 1 GAPDH 0.98 eIF-4α 0.005 PP2A 1.03 3.23
2 eIF-4α 0.98 GAPDH 0.006 SAND 1.07 3.24
3 ACTIN 1.10 TIP41 0.007 TUB 1.14 3.56
4 EF-1α 1.14 ACTIN 0.007 TIP41 1.37 4.09
5 TUB 1.29 UBQ 0.009 GAPDH 1.41 5.05
6 TIP41 1.39 EF-1α 0.011 eIF-4α 1.58 5.17
7 UBQ 1.45 PP2A 0.012 UBQ 1.59 5.26
8 SAND 1.59 TUB 0.015 ACTIN 1.83 6.37
9 PP2A 1.70 SAND 0.017 EF-1α 2.05 7.84
Total 1 ACTIN 0.85 ACTIN 0.012 PP2A 1.24 3.95
2 EF-1α 0.85 TIP41 0.014 TUB 1.45 4.64
3 GAPDH 1.06 eIF-4α 0.021 SAND 1.45 4.49
4 UBQ 1.16 PP2A 0.022 GAPDH 1.62 6.04
5 TUB 1.29 SAND 0.029 TIP41 1.69 5.13
6 eIF-4α 1.37 TUB 0.033 UBQ 1.74 6.02
7 TIP41 1.49 UBQ 0.044 eIF-4α 1.86 6.27
8 SAND 1.59 EF-1α 0.044 ACTIN 1.93 7.10
9 PP2A 1.65 GAPDH 0.048 EF-1α 1.99 8.12
Thereferencegenestabilityincarrotwasanalyzed.Threegroupswereformed:abioticstressgroup(heat,cold,salt,anddrought);hormonestimuli(SA,
GA,MeJA,andABA);andtotal(allsamples).
doi:10.1371/journal.pone.0117569.t002
conditionwereassessedbyusingsixcandidatereferencegenes.Whenthetwomoststable
referencegenesACTINandTUBwereusedfornormalization,theexpressionlevelsof
DcDREB-A1peakedat1handsubsequentlydecreasedat2and4h(Fig.4).Whenthelesssta-
blereferencegeneUBQandEF-1αwereusedfornormalization,similarexpressionpatterns
weregenerated.Bycontrast,whenPP2Awereusedfornormalization,thetranscriptlevelsand
expressionpatternsdifferedfromthoseobtainedusingACTINandothersuitablereference
genes.WhennormalizationwasconductedbasedontheleaststablereferencegenePP2A,the
expressionpatternsofDcDREB-A1peakedat2handdecreasedat4h.
Discussion
qPCRisbroadlyacceptedasamethodwithhighsensitivityandspecificity.Suchmethodis
usedbecauseofitsrepeatedquantitativedynamicrangeandthehigh-throughputanalysisof
PLOSONE|DOI:10.1371/journal.pone.0117569 February6,2015 9/16
ReferenceGenesinCarrotLeaves
Fig4.RelativequantificationofDcDREB-A1geneexpressionnormalizedusingcandidatereferencegenesunderheattreatmentincarrot.
doi:10.1371/journal.pone.0117569.g004
genetranscriptlevels.Accuratenormalizationofgeneexpressionagainstanappropriateinter-
nalcontrolisrequiredforavalidqPCRanalysis.Genetranscriptswithinvariantabundance
undervariousenvironmentalstimuliareessentialreferencepointsforaccuratedataanalysis
[7].Thus,referencegenesshouldbevalidatedundercertainexperimentalconditionsandin
differentspecies[60,64].
Leavesserveimportantfunctionsinprocessofphotosynthesis.Inthegrowthanddevelop-
mentofcarrot,theaccumulatedphotosyntheticproductsweretransportedtotuberousroots.
Furthermore,leafisavitalorganoftheresponsetotheabioticstressandhormonesignal.In
thisstudy,wetestedsuitablereferencegenesfortheexpressionoftargetgenesincarrotleaves.
Ninegenes(GAPDH,ACTIN,eIF-4α,PP2A,SAND,TIP41,UBQ,EF-1α,andTUB)wereselect-
edascandidatereferencegenesforstableexpressionassessmenttestsincarrotleaves.Allnine
candidategeneswereclonedfromcarrotinthisstudybasedonourtranscriptomeandgenome
database,CarrotDB[54].Asinglepeakinthemeltingcurveanalysesconfirmedtheprimer
pairthatshowedspecificity.Plantsweresubjectedtodifferenthormonestimuli(GA,SA,
MeJA,andABA),abioticstresses(heat,cold,salt,anddrought),andefficacydilutions(10×,
102×,103×,104×,105×,and106×dilutions).TheexpressiondatawerecollectedfollowingqPCR
amplificationanddetection.Thecurvesshowedagoodlinearrelationshipdefaultlimitwith
R2>0.99,andtheiramplificationefficienciesrangedfrom93.8%to108.8%(Table1).The
primerpairsandamplificationconditionswereacceptableinqPCR-basedquantification[55].
MostoftheCqvalueswerelyingbetween18and35acrossalltestedsamples,andthemeanCq
valuesrangedfrom24to33[12,60,65,66].Here,theCqvaluesofPP2A(Cq —Cq <10cycles;
max min
PLOSONE|DOI:10.1371/journal.pone.0117569 February6,2015 10/16