4. Designing and validating primers - St. Michael's...
Transcript of 4. Designing and validating primers - St. Michael's...
4.DesigningandvalidatingprimersDESIGN:Whendesigningprimers,considerationshouldbegiventotheareaofthetargetgenebeingamplified(targetsequence)andthethermodynamicpropertiesoftheprimersthemselves.Allofthesepropertiesfactorintotheefficiencyandspecificityoftargetgeneamplification.ForwardandreverseprimerpairsforqPCRshouldamplifyuniquetargetsequencesbetween70-150bplong,thatareareasoflowsecondarystructure(GCcontent<60%).Theprimersthemselvesshouldfollowtheguidelinesforgoodprimerdesign,butbriefly,theirlengthistypically15-20bplong,theirGCcontentshouldbeapproximately50-55%withlowsecondarystructure(hairpins,loopsetc..)butprimersshouldbe“anchored”withaG/Catthe5’endofthesequenceandtheirmeltingtemperatureisapproximately5°Chigherthantheannealingtemperature(60°C)inyourproposedqPCRrunprofile(therefore,optimally65°C).KeepinmindthatalltheprimersusedinasingleqPCRreactionshouldhavesimilarannealing/extensiontemperatures.Therearemanygoodon-linesitesforprimerdesign:PrimerBLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/Primer3 http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/IDTSciTools http://www.idtdna.com/pages/scitools
Sampleread-outfrom“PrimerBLAST”primerdesignprogramITISCRITICALTO“BLAST”(nucleotide)THEPROPOSEDPRIMERSEQUENCETOENSURESPECIFICITYFORTHETARGET(amplified)GENE.Onceyourprimersarrive,iftheyarelyophilized,itisrecommendedtomakea100uMstockandmakeaworkingstock(10uM)fromthis.The100uMstockshouldbekeptat-20°Candopenedonlytocreatenewworkingstocks.Thiswillreducethepossibilityofcontamination.VALIDATION:Itisrecommendedtopurchasethetoptwopredictedprimerpairsandperformthevalidationonbothprimers.Dependingontheavailabilityofyourtemplate(i.e.whetheryoursamplesarereadilyobtainedfromexperimentalanimalsorcellcultureorwhethertheyareprecioushumansamples)youmaywishtovalidateyourprimersonacommerciallyavailableuniversalcDNA.Forexample,forhumanprimersyoucanvalidateonClontechLaboratoriesHumanUniversalReference
TotalRNA(cat#639653/54).WhiletheremaybesomevariabilityinyourtargetgeneexpressionbetweenthiscDNAandthatobtainedfromyourbiopsy/sample,itis“asgoodasyoucanget”withoutwastingyourprecioussamplesonvalidation.
Ideally,themultipleprimersyouwilluseforqPCRallhaveareasonableefficiencyatthesameannealing/extensiontemperature(usually60°C).Youwillhavefactoredthisintothedesignoftheprimerbyspecifyingmelttemperatures(between63-68°C)asoneofthevariables.BeforeperformingaqPCRreactionandusingexpensiveSYBRMasterMix,itisrecommendedtofirstcheckthespecificityofyourprimersbyrunninganendpointPCRreactionfirstandrunningtheproductsonaDNAacrylamidegel.Inthisreaction,youshouldincludeano-templatecontrol(NTC)totestforcontaminationofyourbuffersandsolutionsaswellastheprevalenceofprimer-dimerformation.YoushouldalsoperformmultiplePCRreactionstodeterminetheoptimalconcentrationofprimerstouseforamplification(usuallybetween100-500nM). Sampleend-pointreaction:(50ulfinalvolume)
40ul UPW(ultrapurewater)1ul forwardprimer(10uMstock)1ul reverseprimer(10uMstock)5ul 10XTaqbuffer(whichincludesMgCl2)1ul dNTPmix(2.5mMstock)1ul template(UniversalcDNAortargetcDNA–10-50ng/ulstock)(ORWATER,IFA
NO-TEMPLATECONTROL)1ul Taqpolymerase
PCRParameters:
95oC 2:00minutes
40cyclesof:
95oC 60seconds(denature)
60oC 30seconds(anneal)
4oC hold
RunproductsonanacrylamideDNAgel,preparedasfollows:
8.10mlUltrapureH2O
4.17ml29:1Acrylamide:Bis(30%;Bioradcat#1610156)
0.25ml50xTAEsolution
62.5μlAPS
6.25μlTEMED
Using a SDS-PAGE gel apparatus (i.e. vertical electrophoresis) set up plates and fill entirely with thissolution (i.e., there is no stacking/separating gel). Top with either 10 or 15 well comb. Allowpolymerization,removecomb, flushwellswithwaterand loadPCRsamplewith6X loadingbuffer (forDNA agarose gels NOT sample buffer for protein gels) diluted to 1X. Load DNA ladder according topredictedsizeofproducts.Thegelrequires30-45minutestorun(at120V).Whenlowerloadingdyeis¾of thewaydown thegel, remove fromplatesand incubategel inethidiumbromidebath (250mlH2Owith5μlEthidiumBromidestocksolution(10mg/ml; Invitrogencat#15585-011)) forupto60minutesandthenwashwithwaterforupto60minutes.
*
Figure2.End-pointPCRwithreferencecDNAtoconfirmampliconsize(A)ReferencecDNA(universalhumancDNA)wasutilizedastemplateforend-pointPCRtoverifytheabilityofprimerstoamplifyaSINGLEampliconofpredictedsizeaswellasnobackground(*)incDNAnegativecontrol.
Onceyouhaveconfirmedthatyourprimersrenderasingleproductwithnobackgroundandthe
productmigratesatthepredictedsize,youcanusethisprimersetinaqPCRreactionformelt-curveanalysisandgeneratingastandardcurvetoevaluatePCRefficiency.
Initially,tryonlyafewprimers(i.e.housekeeperssuchasGAPDHandHMBS)togetusedtotheequipment,proceduresandpipetting. It’snotdifficultonceyougetusedto it,butpipettingafull384wellplateisnottrivialforabeginner.It isrecommendedtousea6pointstandardcurve,using1:5or1:10 serial dilutions (in triplicate) of your cDNA template along with NTC and NRT (no reversetranscription)controls,eachintriplicate.
The total reaction volume per well is 10μl (8 ul master mix plus 2 ul template), following a similarformulaforthisSAMPLEofmastermixcalculations:
Negativ
100bp 1 2 3 4 5 6 7 8 9
*Negative Controls (no cDNA)
Reagent [Stock] Volume/rxn samples MasterVol. Finalconcentration
SYBRGreenMix 2x 5.00μl 21 105μl 1x
Primermix(FWD/REV) 10μM 0.40μl 21 8.4μl 400nM(i.e.determinedprior)
H2O(RNase/DNasefree) 2.60μl 21 54.6μl N/A
Total: 8.00μl 168μl
Forserialtemplate(cDNA)dilutions:CommercialhumancDNA(Clontechcat#636693)orexperimentalcDNAwasseriallydiluted1:10enoughfor6wells(perconcentration):i.e.2μlofstockcDNAwasdilutedin18μlH2Oandthenseriallyinthesamemanner(6times)to1:1,000,000.Eachconcentrationwasdonein triplicate foreachprimer2μlof thecDNAwasdispensed intoeachwell. Ofnote,whendispensingintothemicroplate,thepipettetipshouldbeplacedonthesideofthewell: theplatewillbespuntobringeverythingtogether.ThisisaSAMPLEofwhatthe384wellplatewilllooklike.
Mastermixandtemplatearedispensedwitharepeatelectronicpipettor(foraccuracy)intoa384-wellplate. Once the plate is loaded, it must be sealed with an optical cover (ABI4311971) to preventevaporativeloss.Spintheplatefor2minutesat1200rpmandthenruntheqPCRreaction.
Themelt-curveperformedattheendoftheqPCRcyclesisimportantinconfirmingthespecificityoftheprimerannealing.Thecurveshoulddisplayasinglepeakwithnoshouldering(seebelow).
ThestandardcurveisusedtocalculatetheefficiencyoftheqPCRreaction,orhowwellthepolymeraseisabletoamplifythetargetcDNAgiventheprimersequencesandthereactionconditions.Inaperfectlyefficient(100%)reaction,thereisaproductdoublingeverycycle,butreactionefficienciescanbeeitherhigherorlowerthan100%duetoinhibitoryfactorsinthecDNApreparation,ineffectiveprimerbinding,secondarystructureinthetargetgene,ampliconorprimers,oldreagentsandenzymeorsub-optimalannealing/extensiontemperatures.Thestandardcurvewillalsoinformyouoftheoptimaltemplateconcentrationtousesothatyourtargetgenesamplifywithintheoptimalrange(Ct18-25).
Slope:-3.566Y-inter:22.282R2:0.992Eff%:90.737
Theefficiencyiscalculatedbyplottingthetemplatequantityvs.theCtvalue.Theslopeofthe(linearregression)bestfitlinereflectstheefficiencyofthereaction.Thereactionefficiency,shouldalwaysbebetween90-110%anditisimportantthattheefficienciesofalltheprimersusedintheqPCRreactionareapproximatelyequalforaccuratecomparison.TheR2valuerepresentshowwellthedatafittheregressionlineorwhethertheefficienciesaredifferentatanypointintherangeoftemplateconcentrations.ThisR2valueshouldbeascloseto1.0aspossiblewithanacceptablerangebetween0.98-1.0.Ifthevalueissignificantlylower,checkthevariabilitybetweenyourtechnicalreplicatesanddiscardanydatapointsthatfalloutsideof0.5Ctofeachother.Forexample,withtheplotabove,thetriplicatesforthehighestconcentrationofcDNA(22.174,22.471,24.349)wereassessedandtheoutlier(24.349)wasomittedfromtheanalysis.Forstandardcurvesinwhicha1:10dilutionisperformed,thehighestandlowestconcentrationsofthestandardcurvemayhavetobeeliminatedforaccuracyastheCtvaluesmaystart“fallingoffthecurve”withincreasingdlution.Forexample,takethisvalidationscenariousingaGAPDHprimer:
Assessment: (i)optimaldilutionofcDNA(Ct18-25)withfirst3dilutions
(ii)Firstfoursetsofreplicatesaregood,butthenvariabilityishighwhencDNAistoodilute
Canrepeatexperimentanddostandardcurvewithlowerdilution(1:5)tobewithindynamicrange
(iii)Calculatedefficiency(79%)isborderlineacceptable;shouldbe80-110%
(iv)R2=0.998whichisgoodcorrelation
(v)Meltcurvegood–singleproduct,noshouldering
Onceyourprimershavebeenvalidatedforefficiency,R2,andspecificity,youMUSTensurethatyouselectaproperhousekeepinggene(s)foryourexperimentalsystem.Again,referencegenesnormalizethedatabycorrectingfordifferencesinstartingquantitiesofcDNAandtherefore,CANNOTCHANGEINEXPRESSIONLEVEL(CtVALUE)BETWEENEXPERIMENTALCONDITIONS/TIMEPOINTS.Thefinalvalidationstep,therefore,istocomparetheCtvalueatagivenconcentrationofcDNAacrossallyourdifferent
Slope: -3.953 R2: 0.998 Efficiency: 79%
experimentalconditions.Itisalsousefultonotethatoften,onehousekeepinggeneisnotenoughforaccuratedeterminationofgeneexpressionchanges.Vandesompeleetal.(2002)wroteanexcellentarticleoutliningthatthegeometricmeanofmultiplecarefullyselectedhousekeepinggeneswasthemostaccuratenormalizationfactor.Therearealsomanygoodon-linealgorithmstoassessthevariabilityinyourhousekeepersexpressionlevelsforpropernormalization;thesecanbefoundontheGeneQuantificationwebsite(http://www.gene-quantification.info/).
Forpre-validatedhuman,mouseandrathousekeeper(i.e.GAPDH,HMBS,HPRT,CyclophilinA,G6PD)primersequencesseeAppendixIbeloworvisitthefollowingsiteshostedattheUniversityofGhentandHarvard(http://medgen.ugent.be/rtprimerdb/,http://pga.mgh.harvard.edu/primerbank/).Remember,youitisstillagoodideatovalidatethese“pre-validated”primersinyourexperimentalsystembeforeyouusethem!
AppendixI:ValidatedHousekeepingGenesUsedforqPCR(withaccessionnumbers)
•ALWAYS“BLAST”(nucleotide)yourprimersequencesbeforeorderingtocheck!)
•AlsocheckformorevalidatedqPCRprimersin:http://pga.mgh.harvard.edu/primerbank/index.html
HUMAN:
hGAPDH(Glyceraldehyde-3-phosphatedehydrogenase-NM_002046):
Forward5’-CAATGACCCCTTCATTGACC-3’
Reverse5’-GACAAGCTTCCCGTTCTCAG-3’
hG6PD(Glucose-6-phosphatedehydrogenase-NM_000402.3):
Forward5’-GAGGCCGTGTACACCAAGAT-3’
Reverse5’-TCAGGGAGCTTCACGTTCTT-3’
hHMBS(Hydroxymethylbilanesynthase-BC019323.1):
Forward5’-TGCAACGGCGGAAGAAAA-3’
Reverse5’-AGCTGGCTCTTGCGGGTAC-3’
hHPRT1(Hypoxanthineguaninephosphoribosyltransferase1-NM_000194):
Forward5’–CCTGGCGTCGTGATTAGTGAT-3’
Reverse5’–AGACGTTCAGTCCTGTCCATAA-3’
MOUSE:
mGAPDH(Glyceraldehyde-3-phosphatedehydrogenase-NM008084.2):
Forward5’-AGGTCGGTGTGAACGGATTTG-3’
Reverse5’-TGTAGACCATGTAGTTGAGGTCA-3’
mG6PD(Glucose-6-phosphatedehydrogenase-NM008062):
Forward5’–CACAGTGGACGACATCCGAAA–3’
Reverse5’-AGCTACATAGGAATTACGGGCAA-3’
mHMBS(Hydroxymethylbilanesynthase-NM013551.2):
Forward5’-AAGGGCTTTTCTGAGGCACC–3’
Reverse5’-AGTTGCCCATCTTTCATCACTG-3’
mHPRT1(Hypoxanthineguaninephosphoribosyltransferase1-NM_013556):
Forward5’–TCAGTCAACGGGGGACATAAA–3’
Reverse5’–GGGGCTGTACTGCTTAACCAG–3
RAT:
rGAPDH(Glyceraldehyde-3-phosphatedehydrogenase–AF_106860):
Forward5’-GTGCAGTGCCAGCCTCGTC–3’
Reverse5’-GGCAGCACCAGTGGATGCAG–3’
rHPRT(Hypoxanthineguaninephosphoribosyltransferase1-XM_343829):
Forward5’-GCCGACCGGTTCTGTCAT–3’
Reverse5’-TCATAACCTGGTTCATCATCACTAATC–3’
rCycloB(PeptidylprolylisomeraseB(cyclophilinB)-NM_022536):
Forward5’-GGGCTCCGTTGTCTTCCTTT–3’
Reverse5’-GACTTTAGGTCCCTTCTTCTTATCGTT–3’
rHMBS(Hydroxymethylbilanesynthase-NM_013168):
Forward5’-GGCTCAGATAGCATGCAAGAGA–3’
Reverse5’-TGGACCATCTTCTTGCTGAACA–3’