Accurate RT-qPCR gene expression analysis on cell culture lysates.

0.95 MB·English

by  Van Peer, Gert

Accurate RT-qPCR gene expression
analysis on cell culture lysates
SUBJECTAREAS:
GertVanPeer,PieterMestdagh&JoVandesompele
METHODS
GENETICS
CenterforMedicalGenetics,GhentUniversityHospital,9000Ghent,Belgium.
MEDICALRESEARCH
MOLECULARBIOLOGY
Geneexpressionquantificationonculturedcellsusingthereversetranscriptionquantitativepolymerase
chainreaction(RT-qPCR)typicallyinvolvesanRNApurificationstepthatlimitssampleprocessing
Received throughputandprecludesparallelanalysisoflargenumbersofsamples.AnapproachinwhichcDNA
21November2011 synthesisiscarriedoutoncrudecelllysatesinsteadofonpurifiedRNAsamplescanofferafastand
straightforwardalternative.Here,weevaluatesuchanapproach,benchmarkingAmbion’sCells-to-CTkit
Accepted withtheclassicworkflowofRNApurificationandcDNAsynthesis,anddemonstrateitsgoodaccuracyand
23December2011 superiorsensitivity.
Published
13January2012 Geneexpressionquantificationonculturedcellsusingthereversetranscriptionquantitativepolymerase
chainreaction(RT-qPCR)isanimportanttoolinmolecularbiologicalandbiomedicalresearch.Invitro
functional analysis through gene expression measurements can serve as a readout for qualitative or
quantitativeassessmentoftheeffectofdiversetreatmentssuchasRNAianddrugcompoundadministration.
Correspondenceand
Goingfromcellculturestogeneexpressionresultstypicallyinvolvesharvestingofcells,isolationofRNA,removal
requestsformaterials
ofco-purifiedcontaminatingDNAthroughDNasetreatment,cDNAsynthesisandfinallyqPCR.Cellharvesting
shouldbeaddressedto andRNAisolationaretheratelimitingstepsinthisworkflow.Thelimitedthroughputofharvestingprocedures
J.V.(joke. andclassicalRNAextractionmethods-eitherphenol/chloroformbasedRNAextractionandethanolprecipita-
vandesompele@ugent. tionorcolumnbasedsolidphaseextraction-characterizedbytimeconsumingandlaboriousprotocols,preclude
be) theinclusionoflargenumbersofsamplesinasinglecellcultureexperiment.However,thedemandforhigh-
throughputgeneexpressionanalysesoncellculturesisdramaticallyincreasing,amongstothersbyapplicationof
genomewideRNAiandcompoundlibraryscreening.
Toaccommodatethisneed,anelegantmethodwasintroducedforhigh-throughputsynthesisofcDNAtobe
used for qPCR geneexpression analyses of 96-and 384-well cell culture samples, excludingtheneed for cell
harvestingandRNApurification.Themethodusescrudecelllysatesasinputforreversetranscription,signifi-
cantly increasing processing speed and throughput, and offering possibilities towards automation. Different
kits,suchastheCells-to-CTkit(Ambion), theRealTimereadyCellLysiskit(Roche)andtheCellsDirectkit
(Invitrogen),areavailableonthemarket,allofthemcompatiblewithbothDNAbindingdyeorhydrolysisprobe
qPCRdetectionchemistry.Apartfromahandfulreportsonthesensitivity,specificityandcompatibilitywith
automation1,2,littleisknownabouttheperformanceintermsofaccuracyofthisnewmethod.Here,weperformed
an extensive characterization of the workflow by evaluating the Cells-to-CT kit, and benchmarked it to the
hithertogoldstandardworkflowofRNApurificationandcDNAsynthesis.Wedemonstratesuperiorsensitivity,
good accuracy, and effective DNase treatment when using crude cell lysates. Both low and high-throughput
RT-qPCRexperimentsoncellsgrowninmicrotiterplatesmaysubstantiallybenefitfromthisnewworkflow.
Results
Accuracy. First, the abilityof the Cells-to-CT workflow to accurately quantify relative gene expression levels
andresultingfoldchangesbetweensampleswasassessed.Tothisend,cDNAfromduplicateculturesoffour
neuroblastomacelllines,twobearingamplificationoftheMYCNtranscriptionfactorgene(NGPandIMR-32)
andtwowithouttheamplification(SH-EP,SK-N-AS),waspreparedusingeithertheclassicortheCells-to-CT
workflow. Relative expression levels of 10 genes of interest (DKK3, INHBA, PLAT, RGS4, MYC, MTHFD2,
MYCN,TGFBI,PMP22,NTRK2)knowntobedifferentiallyexpressedbetweencellswithandwithoutMYCN-
amplification3werethenquantified,yieldingatheoreticalnumberof80expressionleveldatapointsforboth
workflows.Thefractionofmissingdatawaslowandcomparableforbothmethodsandoccurredforgeneswith
verylowexpression,asexpected.FortheclassicandtheCells-to-CTworkflow,respectively78and76relative
expressionlevelscouldbeeffectivelydetermined.Foldchangeswerecalculatedamongallcelllines(28paired
SCIENTIFICREPORTS |2:222|DOI:10.1038/srep00222 1
www.nature.com/scientificreports
comparisons for 4 cell lines in duplicate). Theoretically 280 fold (p-value Fisher’s Exact Test , 0.001; Fig. 2c), in high log fold
2
changes could be calculated, and when taking into account the changes(suppl.Table1)andinlog foldchangesforspecificqPCR
2
missing data, 266 and 254 fold changes could be effectively cal- assays (suppl. Table 2). High Cq value was the only independent
culated for the classic and the Cells-to-CT workflow, respectively. predictorvariableofsuboptimalconcordanceinthisdataset.
Theassessmentoffourdifferentcelllinesandtengenesresultsina
widecoverageoffoldchanges,rangingfromnochangetomorethan Sensitivity and linearity. In order to compare the sensitivity of
athousandfoldchange.Relativeexpressionlevelsandfoldchanges working with cell lysates versus working with purified RNA as
werelog transformedforfurtherdata-analysis. input for reverse transcription, a cell dilution series of the neuro-
2
HighandsignificantcorrelationscouldbeobservedbetweenCq blastomacelllineSH-EP(20,000,10,000,5,000and2,500cells)with
values (Fig. 1b, Pearson r 5 98%; Spearman r 5 97%), log nor- duplicate samples was either subjected to classic RNA isolation
2
malizedrelativequantities(Fig.1a,b&suppl.Fig.1,Pearsonr596%, immediately after dilution series preparation or seeded in 96-well
Spearmanr598%)andlog foldchanges(Fig.1b,Pearsonr599%, cellculture plates and lysedwithCells-to-CT reagents after6 h of
2
Spearman r 5 98%) obtained with both methods. A cumulative adherence. An approach like this, with immediate RNA isolation
distribution plot of the difference in log fold change determined andquasiimmediateCells-to-CTcelllysis,guaranteesminimaldif-
2
with both methods (Dlog fold change) shows that this difference ferencesincellinputtobothmethods,asadherencetimeisbelowthe
2
issmallerthan0.5formorethan63%,smallerthan1formorethan doubling time of SH-EP cells, and at the same time meets the
83% and smaller than 1.5 for more than 93% of the fold changes requirementtohavecompletelyadheredcellsincellcultureplates
observed(Fig.2a).SimilarconclusionscanbedrawnfromaBland- forCells-to-CTlysatepreparation.
Altmanplotforthelog foldchanges(Fig.2b),witha95%confidence As raw Cq values, rather than normalized relative expression
2
intervalofthetruedifferencethatrangesfrom1.57to21.76.Bland- levels,arethesubjectofcomparisonherewherebynonormalization
Altman plots are often regarded as better measures to assess the occurs,ideallyreversetranscriptiononbothRNAsamplesandcell
agreementoftwomethodsthansimplecorrelationanalyses. lysatesisperformedinthesamerunandwiththesameRTproced-
Log fold changes that showed suboptimal concordance, here ure.Therefore,theCells-to-CTreversetranscriptionkit,whichcan
2
definedashavingadeltalog foldchangegreaterthan1,weresig- copewithbothtypesofinput,wasusedonbothpurifiedRNAaswell
2
nificantlyenrichedinfoldchangescalculatedfromhighCqvalues ascelllysates.TonormalizethecDNAinputfortheqPCRreactions
Figure1|Accuracy. (a)Log normalizedrelativequantitiesquantifiedwitheithertheclassicworkflow(black)ortheCells-to-CTworkflow(grey)for6
2
genes(DKK3,INHBA,PLAT,RGS4,MYC,MTHFD2).(b)CorrelationbetweenCqvalues,log normalizedrelativequantitiesandlog foldchanges
2 2
quantifiedwitheithertheclassicortheCells-to-CTworkflow.
SCIENTIFICREPORTS |2:222|DOI:10.1038/srep00222 2
www.nature.com/scientificreports
Figure2|Accuracy. (a)CumulativedistributionofDlog foldchanges.(b)Bland-Altmanplotforlog foldchanges.Thedifferenceinlog foldchange
2 2 2
measuredwithbothmethodsisplottedagainsttheaverageoftheselog foldchanges.The95%confidenceintervalofthetruedifferenceisdelineatedby
2
theupper(ULA)andlowerlimitofagreement(ULA).(c)Enrichmentanalysis.EachdatapointinthecumulativedistributionplotofDlog foldchanges
2
resultsfromtwolog foldchanges,eachcalculatedfromtwolog relativequantitiesthatbothinturnresultfromfiveCqvalues(onegeneofinterest,and
2 2
fourreferencegenes).Datapointsinpurplearedatapointsforwhichatleast1ofthe20Cqvaluesgivingrisetothisdatapointishigherthanthespecified
cutoff.
totheinputcellnumberusedforRNAisolationorCells-to-CTlysis, Discussion
equivalentfractionsofeithertotalRNAisolateorcelllysate(18%of Cell harvesting and RNA purification are the rate limiting steps
totalvolume)wereusedasinputforreversetranscription.qPCRwas in going from cell cultures to cDNA and thus eventually to gene
performedforfourgenes(SDHA,HPRT1,GAPDH,UBC).Toavoid expression levels andfoldchanges usingRT-qPCR. Analternative
theintroductionofinter-runvariation,allqPCRreactionswereper- method for cDNA synthesis that eliminates these steps and uses
formedinthesamerunusingthesamemastermixandPCRcondi- crude cell lysates instead of purified RNA as an input for reverse
tions(Eurogentecprotocol). transcriptioncouldofferafastandstraightforwardalternative,sig-
Foreverypointinthecelldilutionseriesandforeverygenetested nificantlyincreasingsampleprocessingthroughput.Here,weshow
theCqvalueswereonaverage4cyclesearlierwhenusingcelllysates for the first time the reliability and accuracy of this approach by
(Fig.3),clearlyillustratingthesuperiorsensitivityoverworkingwith extensivelyevaluatingoneofthecommerciallyavailablekits.Other
purifiedRNAfractions.Furthermore,justasforclassicRNAisola- kits on the market should undergo the same level of performance
tion,withinarangeofcellnumbersfrequentlyusedforseedingin96- assessment prior to their routine use, following the testing frame-
wellcellcultureexperiments,cDNAyieldswerelinear. workpresentedhere.
We clearly observed high concordance between RT-qPCR gene
DNasetreatment.ToevaluatetheperformanceofDNasetreatment expressionresultscomingfromcelllysatesorpurifiedRNAasinput
oncrude cell lysates, single replicate Cells-to-CT lysates were pre- forthereversetranscription,andthisoverawiderangeofexpression
paredbothinthepresenceandtheabsenceofDNaseIinthelysis differences measured for various genes in multiple cell lines. For
buffer for four neuroblastoma cell lines (SH-EP, SK-N-AS, NGP, normalizedrelativeexpressionlevelsandfoldchanges,boththecor-
IMR-32). The crude cell lysates were directly used as input for relationandtheagreementofvalueswashigh,demonstratingthat
qPCRusingfourDNAspecificassays(NEUROD1,XRCC3,PLAT, bothmethodscouldbeusedinterchangeablywithgreatconfidence.
MTHFD2). Due to a combination of a difference in sensitivity and in input
DNasetreatmentappearedtobeefficient.Acompleteoralmost amounts into qPCR reactions between both workflows for cDNA
completeeradicationoftheDNAspecificsignalcouldbeobserved synthesis, raw Cq values did not show a high agreement. Rather,
withallassaysinallcelllines(Fig.4). there was a constant and systematic bias that could be eliminated
SCIENTIFICREPORTS |2:222|DOI:10.1038/srep00222 3
www.nature.com/scientificreports
Figure3.|Sensitivityandlinearity. Cqvaluesforfourgenesquantifiedonacelldilutionserieswitheitherthegoldstandardworkflow(grey)orthe
Cells-to-CTworkflow(black).
throughnormalization.Inthisdataset,only17%ofthefoldchanges incomplete4,soitisprobablynotrelatedtothecomplexenvironment
would have been differentially called by more than a factor 2 (i.e. of a crude cell lysate. Incomplete DNase digestion potentially also
a log fold difference of 1) when applying one method or the partially explains the discrepancies observed for log fold changes
2 2
other.However,asthis17%almostexclusivelyconsistsoflargefold involvinghighCqvaluesandthuslowlyexpressedgenes.
changes,thisdoesnotposeaseriousproblem,astheexactmagnitude Apartfromtheimprovementsintermsofspeed,throughputand
ofthefoldchangeisoftenoflessimportance.Inaddition,the17% easeofsamplehandling, anothermain advantage ofworking with
suboptimallycorrelatingresultsarealsohighlyenrichedinlog fold celllysatesisitsbettercompatibilitywiththesmallercellcultureplate
2
changesresultingfromhighCqvalues.InherenttoqPCR,thevari- formats(i.e.96-wellandeven384-wellformat).Togetherwiththe
ationincreaseswithhigherCqvaluesandtheresultingfoldchanges highersensitivitythisenablessubstantialdownscalingofreactions,
arethuslessaccurateandprecise.Hence,thediscrepanciesinlog herebyloweringreagentusageandoverallexperimentcost.Inaddi-
2
foldchangesobservedaremostlikelynotrelatedtotheinaccuracyof tion, together with the fact that cell harvesting is not required
onemethodortheother,butduetothisphenomenoninherentto anymore, it allows total automation of cDNA synthesis from cell
qPCR.Itshouldbenotedthatthelevelofagreementbetweenboth cultures1. Classic phenol/chloroform extraction is not amendable
methods observed is in fact an underestimation of their true con- toautomation.OtherconventionalmethodsforRNAisolationhave
cordance, as the nature of the techniques makes it impossible to beenadaptedtohigh-throughputplatforms.Examplesaretheuseof
execute both workflows on the same cell culture sample and thus glassfiberfilterplateswhereRNAbindstothefilterinthepresenceof
correlated data obtained here originate from cell culture replicates highsaltandalcoholconcentrationsortheuseofmagneticmicro-
culturedindifferentplateformats(i.e.6-wellfortheclassicworkflow sphericbeadstoselectivelybindnucleicacids.However,thesemeth-
usingpurifiedRNAand96-wellplatesfortheCells-to-CTworkflow). odsstillrequireagreatdealofuserinteraction.
Next to a good accuracy, a higher sensitivity for the lysate ap- In conclusion, we here provide solid evidence for the accuracy
proach compared to the classic workflow could be demonstrated. ofRT-qPCRgeneexpressionquantificationusingcelllysatesasan
Thismaybeexplainedbythefactthatfewerstepsandnopurification input for reverse transcription, along with a confirmation of the
are required when working with cell lysates, meaning there is less superiorsensitivityofthisapproach.
chancetointroducebiasinagivensample’smRNAcontent.
Finally,wepositivelyevaluatedtheperformanceofDNasetreat-
Methods
ment on crude cell lysates, with either a complete or an almost Theproceduresinthissectiondescribebothaclassicworkflow(RNApurification)
completeremovalofthecontaminatinggenomicDNA.Incomplete andtheCells-to-CTworkflowforgeneexpressionquantification.Specific
digestionofDNAonlyposesaproblemforlowlyexpressedgenes, adaptationsmadetobothworkflowstotestparticularaspectsaredescribedwhere
relevantintheResultssection.
astheremainingDNAspecificsignalmaycontributerelativelymore
tothetotalsignal.LongerDNaseIincubationtimesthantheones
Cellculture.Neuroblastomacelllines(SH-EP,SK-N-AS,NGP,IMR-32)were
proposedbythemanufacturermayfurtherimprovetheefficiencyof
culturedinRPMI-1640(Invitrogen)supplementedwithfetalcalfserum(10%),
thedigestion.Ofnote,DNasedigestiononpurifiedRNAisalsooften L-Glutamine(1%),penicillin/streptomycin(1%),kanamycine(1%)andHEPES
SCIENTIFICREPORTS |2:222|DOI:10.1038/srep00222 4
www.nature.com/scientificreports
Figure4|DNasetreatment. DNAspecificsignalonlysatesfrom4differentcelllinesgeneratedeitherintheabsence(2)orinthepresence(1)of
DNaseI,using4differentDNAspecificqPCRassays.
(25mM).TosynthesizecDNAaccordingtotheclassicworkflow,cellswereseededin 2. Tavares,R.L.C.,Xu,K.,Zhang,C.&Freitas,V.Aspecificandquickgene
6-wellcultureplatesatadensityof250,000cells/well.Alternatively,tosynthesize expressionstudyinmouseEScells.JAssistReprodGenet24,366–372(2007).
cDNAusingtheCells-to-CTkit,cellswereseededin96-wellcultureplatesatadensity 3. Vermeulen,J.etal.RNApre-amplificationenableslarge-scaleRT-qPCRgene-
of10,000cells/well.cDNAsynthesiswascarriedout48haftercellseeding. expressionstudiesonlimitingsampleamounts.BMCResNotes2,235(2009).
4. Tavares,L.,Alves,P.M.,Ferreira,R.B.&Santos,C.N.Comparisonofdifferent
cDNAsynthesis.TheclassicworkflowforcDNAsynthesisconsistsofcellharvesting methodsforDNA-freeRNAisolationfromSK-N-MCneuroblastoma.BMCRes
byscraping,phenol/guanidinethiocyanate-chloroformbasedlysisofcellsin Notes1,140(2011).
combinationwithsilicamembranebasedRNAextractionusingthemiRNeasyMiniKit 5. Bustin,S.A.etal.TheMIQEguidelines:minimuminformationforpublication
(Qiagen),on-columnDNaseIdigestionusingtheRNase-FreeDNaseSet(Qiagen), ofquantitativereal-timePCRexperiments.ClinicalChemistry55,611–622
followedbyoligo(dT)/randomprimermediatedreversetranscriptionusingtheiScript (2009).
cDNASynthesisKit(Bio-Rad)with500ngofRNAasinput.Alternatively,theCells-
6. Lefever,S.,Vandesompele,J.,Speleman,F.&Pattyn,F.RTPrimerDB:theportal
to-CTkit(Ambion)wasusedtoproduceDNaseIdigestedcelllysatesandperform
forreal-timePCRprimersandprobes.NucleicAcidsRes37,D942–D945(2009).
cDNAsynthesis.Allkitswereusedaccordingtomanufacturer’sinstructions.
7. Hellemans,J.,Mortier,G.,DePaepe,A.,Speleman,F.&Vandesompele,J.qBase
relativequantificationframeworkandsoftwareformanagementandautomated
RT-qPCR.RT-qPCRgeneexpressionquantificationswereperformedandreported
analysisofreal-timequantitativePCRdata.GenomeBiol8,R19(2007).
accordingMIQEguidelines5.Allreactionswereperformedin384-wellplatesusingthe
8. Vandesompele,J.etal.Accuratenormalizationofreal-timequantitativeRT-PCR
LightCycler480instrument(Roche).ReactionswithclassiccDNAasinputwere
databygeometricaveragingofmultipleinternalcontrolgenes.GenomeBiol3,
performedinatotalvolumeof8ml,comprising4ml2xSYBRGreenIMasterMix
RESEARCH0034(2002).
(Eurogentec),2.5ngcDNA(totalRNAequivalents)and235nMofeachprimer(final
concentration).ForgeneexpressionanalysisoftheCells-to-CTcDNA,adownscaled
versionoftheqPCRprotocolsuppliedwiththeCells-to-CTkitwasused,withreactions
performedin10ml,comprising5mlPowerSYBRGreenMasterMixsuppliedwiththe
Authorcontribution
kit,1/50thofthecDNAsampleand300nMofeachprimer(finalconcentration).
AllqPCRreactionswereperformedinduplicateandCqvalueswereaveraged. G.V.P.executedtheexperiments,performedtheanalysesanddraftedthemanuscript.P.M.
Liquidhandlingwasdoneusingapipettingrobot(TECANEvo100).AllqPCRassays andJ.V.supervisedthestudy,revisedthemanuscriptandapprovedthefinalversionofthe
(suppl.Table3&4)wereextensivelyvalidatedinsilicousingtheRTPrimerDBprimer manuscript.
evaluationpipeline6andempiricallyvalidated,checkingbothprimerefficiencyand
specificity.Calculationofnormalizedrelativeexpressionlevelswasdoneusingthe
Additionalinformation
qbasePLUSsoftwareversion1.57(Biogazelle).Normalizationwasperformedusingfour
stablyexpressedneuroblastomareferencegenes(AluSq,HPRT1,TBPandYWHAZ) Supplementaryinformationaccompaniesthispaperathttp://www.nature.com/
validatedusingthegenormPLUS8moduleinqbasePLUS. scientificreports
Competingfinancialinterest:Theauthorsdeclarenocompetingfinancialinterests.
Statistics.AllstatisticalanalyseswereperformedusingtheRstatisticalprogramming
License:ThisworkislicensedunderaCreativeCommons
environment(version2.10.1).
Attribution-NonCommercial-ShareAlike3.0UnportedLicense.Toviewacopyofthis
license,visithttp://creativecommons.org/licenses/by-nc-sa/3.0/
1. Abruzzese,R.,Fekete,R.&Kephart,D.Cells-to-CTKits:NextGenerationGene Howtocitethisarticle:VanPeer,G.,Mestdagh,P.&Vandesompele,J.AccurateRT-qPCR
ExpressionAnalysisWorkflowsthatEliminateSamplePurification.Journalofthe geneexpressionanalysisoncellculturelysates.Sci.Rep.2,222;DOI:10.1038/srep00222
AssociationforLaboratoryAutomation15,362–368(2010). (2012).
SCIENTIFICREPORTS |2:222|DOI:10.1038/srep00222 5