斑马鱼胚胎qRT-PCR检测应用qRT-PCR of Zebrafish protocols - 图文

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ProtocolQuantitativeReal-TimeRT-PCR(qRT-PCR)ofZebrafishTranscripts:OptimizationofRNAExtraction,QualityControlConsiderations,andDataAnalysis

Chuan-ChingLan,1RongyingTang,1IvoneUnSanLeong,1andDonaldR.Love1,2,3

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SchoolofBiologicalSciences,TheUniversityofAuckland,Auckland1142,NewZealandLabPLUS,AucklandCityHospital,Auckland1148,NewZealand

INTRODUCTION

Thezebrafish(Daniorerio)hasemergedasapopularmodelspecies.Therapiddevelopmentofzebrafishembryosprovidesopportunitiesforinvestigationofgenesessentialfordevelopmentalprocesses,thehumancounterpartsofwhichmightbeimplicatedindiseases.Understandingwhenandwheregenesareexpressedcanfacilitategreaterunderstandingoftheirfunction,andalsoallowthegenestobemanipulatedbygeneknockdownintemporallyandspatiallyspecificmanners.Quantitativereal-timepolymerasechainreaction(qRT-PCR)iswidelyappliedingeneexpressionstud-ies.ThisprotocolpresentstechniquestooptimizeRNAisolationfromzebrafishembryos;qualityassessmentandtheuseofmultiplereferencegenesarealsoemphasized.ThecombineduseofTRIzolextractionandcolumn-basedpurificationisstronglyrecommended,becausetheresultingRNAisofbetterqualitythanRNAisolatedusingeitherofthosemethodsalone.Theprocedurecanbeperformedin2d,withindividualstagestakingupto15htocomplete.

RELATEDINFORMATION

Thisprotocolisadaptedfromthemanufacturers’instructionsfortheuseofTRIzol(Invitrogen)andtheRNeasyMicrokit(QIAGEN).Thecombinedmethodiswidelyusedbyresearchersacrossdifferentdisciplines.However,thisprotocolsuggestsextrastepsthathelpimprovethequalityandquantityofRNAisolatedfromzebrafishembryos.ForanalysisofqRT-PCRdata,softwaresuchasgeNorm(http://medgen.ugent.be/~jvdesomp/genorm;Vandesompeleetal.2002)andLinRegPCR(http://www.hartfaalcentrum.nl/index.php?main=files&sub=0;Ramakersetal.2003;Ruijteretal.2009)isavailablefordownload.

MATERIALSCAUTIONSANDRECIPES:PleaseseeAppendicesforappropriatehandlingofmaterialsmarkedwith,andrecipesforreagentsmarkedwith.Reagentsβ-mercaptoethanol,≥98%(SigmaM3148)AgilentRNA6000NanoKit(AgilentTechnologies5067-1511)ThiskitisdesignedforusewithAgilentTechnologies’2100Bioanalyzer.3Correspondingauthor(DonaldL@adhb.govt.nz).Citeas:ColdSpringHarbProtoc;2009;doi:10.1101/pdb.prot5314www.cshprotocols.org?2009ColdSpringHarborLaboratoryPress1

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ChloroformDiethylpyrocarbonate(DEPC)-treatedH2ODNase,RNase-free(e.g.,RNase-FreeDNaseSet;QIAGEN)dNTPmix(10mM;Invitrogen)(forreversetranscription)Ethanol(100%,80%,and75%,preparedinDEPC-treatedH2O)Glycogen,(20mg/mL;Invitrogen)H2O,DNase-andRNase-freeIsopropanolPlatinumSYBRGreenqPCRSuperMix-UDGwithROX(Invitrogen)Randomprimers(3μg/μL;Invitrogen)Dilutestockto50ng/μLinDEPC-treatedH2Obeforeuse.ReagentsforsequencingampliconsReversetranscriptase(e.g.,SuperScriptIII;Invitrogen)RNAAssayKit,Quant-IT(Invitrogen)(optional;seeStep45)ThiskitisdesignedspecificallyforusewiththeQubitfluorometer.RNaseZap(Ambion)RNeasyMicroKit(QIAGEN)ThekitincludesDNase,RDDbuffer,RLTbuffer,RPEbuffer,RW1buffer,1.5-mLeluatetubes,2-mLcollectiontubes,andMinElutespincolumns.SDS(sodiumdodecylsulfate;10%)Dilutethe10%stockto1%withDEPC-treatedH2Obeforeuse.TRIzol(TRIzol,Invitrogen)ZebrafishembryosofthestageofdevelopmentofinterestEquipmentBioanalyzer(e.g.,2100;AgilentTechnologiesG2940CA)DryiceFastreal-timePCRsystem,equippedwith384-wellblockmoduleandautomationaccessory(e.g.,7900HT;AppliedBiosystems4329002)andsoftware(e.g.SequenceDetectionSystem[SDS]v2.3;AppliedBiosystems)Centrifuge,equippedformultiwellplatesCentrifuge,refrigerated,equippedformicrocentrifugetubesCentrifuge,ventilated,formicrocentrifugetubesEquipmentforsequencingampliconsFluorometer,Qubit(InvitrogenQ32857)(optional;seeStep45)FumehoodGloves,disposableHomogenizer,hand-held(e.g.,PRO200;PROScientific01-02200)IceLiquidnitrogen(optional;seeStep4.i)MicropipettorsOpticaladhesivefilm(e.g.,MicroAmp;AppliedBiosystems)Pipettetips,barrier,10-,20-,200-,and1000-μLPipettetips,filter,50-μL,PCRclean(e.g.,Eppendorf0030003.950)ThesetipsaredesignedforusewiththeepMotionautomatedpipettingsystem.PipettesPipettes,transfer,single-use,3.5-mL(Sarstedt86.1172.001)Pipettingsystem,automated(e.g.,epMotion5075LH,230V;Eppendorf5075000.008)Plates,384-well(e.g.,MicroAmpoptical384-wellreactionplatewithbarcode;AppliedBiosystems)Softwarefordataanalysis(e.g.,geNorm[seeStep51.iii,Step60]andLinRegPCR[seeStep58])(seealsoRelatedInformationandDiscussion)www.cshprotocols.org2

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Softwareforprimerdesign(optional;seeStep50)

Spectrophotometer(e.g.,NanoDrop;ThermoScientific)(optional;seeStep45)ThermalcyclerTimer

Tuberacks,for1.5-to2-mLmicrocentrifugetubes

Tubes,microcentrifuge,clear,1.7-mL(e.g.,AxygenMCT-175-C)

Tubes,PCR,thin-wall,flat-cap,0.2-mL(e.g.,MAXYMumRecovery;AxygenPCR-02-L-C)Tubes,RNase-free,nonstick,1.5-mL(e.g.,AmbionAM12450)

METHOD

TominimizeRNasecontamination,cleanworksurfaceswithRNaseZapthoroughly.Opentubesfacingawayfromtheoperator,andavoidbreathingintotubes.Dispensealiquotsofsolutionsintosteriletubes;disposeoftubesaftereachuse.Changeglovesfrequently,andunlessspecifiedintheprotocol,keepRNAoniceasmuchaspossible.

Preparation

1.Soaktheprobeofthehomogenizerin1%SDSovernight.

2.Washthe1.7-mLmicrocentrifugetubestobeusedforhomogenization:

i.

Washoncewith1mLofDEPC-treatedH2O.

ii.Washoncewith1mLof75%ethanol.iii.Washoncewith800μLofTRIzol.

SampleCollection

Embryodechorionationisnotmandatory,butcanbeperformedifdesired.

3.Dispense20-50embryosintoawashed1.7-mLmicrocentrifugetube.Removeexcessliquidusing

adisposabletransferpipette.

4.Euthanizetheembryosusingoneofthefollowingmethods:

Theeffectsofanesthetics(e.g.,Tricaine)ontheefficiencyoftheextractionprotocolanddownstreamamplifica-tionoftranscriptsarenotknownatpresent.

Freezing:i.

Thisisthepreferredmethodofeuthanasia.

Immersethetubeinliquidnitrogen.Samplescanbestoredat-80°C.ii.ProceedtoStep5.

TRIzolextraction:iii.PrechillTRIzolonice.

iv.Add1mLofTRIzoltoeachtube.

v.ProceedimmediatelytoStep5andthenStep8.

Homogenization

5.Washthehomogenizationprobe(10-seceachwash):

i.

WashtwicewithDEPC-treatedH2O.

ii.WashoncewithRNaseZap.iii.WashoncewithDEPC-treatedH2O.

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iv.Washoncewith75%ethanol.v.WashoncewithTRIzol.

6.Transferfrozenembryosfrom-80°Cstoragetothebenchondryice.

7.Add1mLofTRIzoltoa1.7-mLmicrocentrifugetubecontainingfrozenembryos.8.Homogenizethesamplefor30sec(Fig.1).9.Incubatethetubeondryicefor30sec.

Allowthehomogenizertocooldowntopreventoverheatingofthesample.10.Homogenizethesamplefor30sec.

11.Incubatethesamplefor5minatroomtemperature.

RepeatSteps8-11foreachsample.Betweensamples,washtheprobe,10seceachwash,oncewith75%ethanol,oncewithDEPC-treatedH2O,andoncewithTRIzol.

12.Placesamplesondryiceforsame-dayuse,orstoreforlateruseat-80°C.13.Afterhomogenizingallsamples,cleantheprobe(10seceachwash):

i.

Washoncewith75%ethanol.

ii.WashoncewithDEPC-treatedH2O.iii.Washoncewith75%ethanol.iv.WashoncewithDEPC-treatedH2O.

RNAIsolationwithTRIzol

14.Thawfrozenhomogenizedsamplesonice.

15.Add200μLofchloroformtoeachsample.Shakevigorouslyfor15sec.Incubatefor3minatroom

temperature.

16.Centrifugeat10,000gfor15minat4°C.

17.Transfer550μLoftheaqueousphasetoanRNase-freetube.

18.Add1μLof20-mg/mLglycogentoeachsample.Add550μLofisopropanol.

Foryoungembryosorforasmallnumberofembryos,addingglycogenincreasesRNAyieldssignificantly.19.Incubatefor10minatroomtemperature.

FIGURE1.Homogenizersetupinaportablefumehood.

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