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’