Characterization of specific cDNA background synthesis introduced(1).pdf

Research paperCharacterization of specific cDNA background synthesis introducedby reverse transcription in RT-PCR assaysM.F.Adrovera,M.J.Muozb,M.V.Baeza,J.Thomasc,A.R.Kornblihttb,1,A.L.Epsteinc,1,D.A.Jerusalinskya,*,1aInstituto de Biologa Celular y Neurociencias“Prof.Eduardo De Robertis”IBCN-CONICET-Facultad de Medicina,Universidad de Buenos Aires,Paraguay 2155,2do piso(1121)Ciudad de Buenos Aires,ArgentinabLaboratorio de Fisiologa y Biologa Molecular,Departamento de Fisiologa,Biologa Molecular y Celular,IFIBYNE,CONICET-Facultad de Ciencias Exactas y Naturales,Universidad de Buenos Aires,ArgentinacCentre de Gntique Molculaire et Cellulaire,Universit Claude Bernard Lyon 1,CNRS-UMR5534,Villeurbanne,Francea r t i c l e i n f oArticle history:Received 12 April 2010Accepted 30 July 2010Available online 13 August 2010Keywords:Reverse transcriptionAntisense RNABackground synthesisRT-PCRHSV-1 vectorsa b s t r a c tTo block expression of NMDA receptor NR1 subunit,we injected into rat hippocampus a Herpes SimplexVirus type 1 derived vector bearing a sequence for NR1 antisense.RT-PCR assays with RNA fromhippocampus of animals injected either with NR1 antisense vector,control vector or vehicle,showed anamplification signal compatible with NR1 antisense which could be attributed either to an endogenousNR1 antisense or to an artifact.RT-PCR was performed either with different primers or without primersin the RT,using RNA from different tissues.RNAse protection assay was carried out to characterize theamplified signal nature.Our results show that the template for the unexpected amplified fragment wasNR1 mRNA currently expressed in nervous tissue.We considered this basal amplification of a mRNA ina RT-PCR assay as“background amplification”.After background subtraction,a significant signal onlyremained when samples from NR1 antisense vector injected animals were used,demonstrating that thiswas the only source for NR1 antisense.Background amplification at RT in the absence of primers,canconstitute a troubling factor in quantitative nucleic acid determination,leading to major interference,particularly when both sense and antisense sequences are present in the sample.Since RT introduceda significant background signal for every gene analyzed,we propose that RT must be always performedalso without primers.Then,this signal should be identified,quantified and subtracted from the specificreaction amplification signal.?2010 Elsevier Masson SAS.All rights reserved.1.IntroductionReverse transcription polymerase chain reaction(RT-PCR)is themost common method for characterizing or confirming geneexpression and comparing mRNA levels in different samples 1,2.Furthermore,real-time RT-PCR is a sensitive and accurate tech-nique for mRNA quantification 2.To estimate the expression of a gene,its transcript must be firstcopiedto cDNAby reverse transcription.Primers usedforcDNA synthesis in reverse transcription(RT)assays are either oligo(dT),random oligonucleotides or sequence specific oligonucleo-tides.These are not natural primers for reverse transcriptases.Retroviruses and retrotransposons utilize specific cellular tRNAs asprimers for reverse transcription of their own initiator RNA,resulting in a double stranded cDNA 3e5.On the other hand,endogenous small RNAs could also be effective as primers for cDNAsynthesis 6.Frech and Peterhans(1994)reported that this cDNAcould lead to a background signal in a subsequent PCR assay.Toreduce this“background signal”,cellular RNA was immobilized onmembranes.However,further addition of either specific oligonu-cleotides or total RNA from different origins still served as primersfor cDNA synthesis,resulting in an increase of PCR signal 7.It hadalso been hypothesized that purification of mRNA could preventbackground priming and,therefore,that background signal wouldbe eliminated.However,mRNA preparations obtained usingdifferent purification methods could still be reverse transcribed,even in the absence of any added primer 8.The occurrence of background amplification in the absence ofprimers at the RT reaction can constitute a significant troublingfactor in quantitative nucleic acid determination.Moreover,it couldlead to major interference when intending to detect a specific RNA*Corresponding author.Tel.:54 11 5950 9500 x2219;fax:54 11 5950 9626.E-mail address:(D.A.Jerusalinsky).1Equal contribution to this paper.Contents lists available at ScienceDirectBiochimiejournal homepage: see front matter?2010 Elsevier Masson SAS.All rights reserved.doi:10.1016/j.biochi.2010.07.019Biochimie 92(2010)1839e1846present in both sense and antisense sequences in the same sample.This is relevant,for instance,in the field of virology,where viralRNA can be present as the positive and negative strand 9,10,oreven in any sample where sense/antisense pairs of sequences areexpressed 11e13.In a previous study,we have blocked the expression of NR1subunit of NMDA receptor using herpes simplex virus type 1(HSV-1)-derived amplicon vectors bearing a sequence coding fora NR1 antisense.Then we intended to show NR1 antisense expres-sionfromthevectorinjectedintorathippocampus,wheretheNR1iscurrently expressed 14.However,using total hippocampal RNA,a RT-PCR product compatible with a NR1 antisense was obtainedeven in control animals.In the case of non-infected tissue,thisunexpected fragment could be attributed either to an endogenousNR1 antisense RNA or to an artifact.In order to distinguish betweenthese two possibilities,we carried out diverse RT-PCR assays,eitherwith different primers or without primers in the RT assay.We alsoperformed RNAse protection assay(RPA)in an attempt to charac-terize the nature of the unexpected amplification signal.2.Material and methods2.1.HSV-1-derived amplicon vectorsAmplicon vector stocks were prepared as already described byZaupa et al.(2003),with minor modifications.Briefly,Vero 7b cells(which are modified Vero cells transcomplementing HSV-1 ICP4and ICP27 genes)were maintained in culture in Dulbeccosminimum essential medium(DMEM)(Invitrogen,Paisley,UK)supplemented with inactivated Fetal Bovine Serum 10%(FBS)(Invitrogen),Penicillin(100 U/ml)and Streptomycin(100mg/ml)(Invitrogen).Cell lines were transfected with the amplicon plasmidusing LipofectAMINE Plus Reagent(Invitrogen),according tomanufacturers instructions.One day later,transfected cells wereinfected with helper HSV-1 LaLDJ at a multiplicity of infection of 0.3plaque forming units/cell.When cytopathic effect was maximum,cells were collected by centrifugation and disrupted by three cyclesof freezing and thawing to release vectors particles.Media con-taining amplicon vectors were then centrifuged topellet cell debris.Helper virus particles in the supernatant were titrated by plaqueassay on Vero 7b cells 15.To titrate vector particles,Gli36 cells16 were infected with serial dilutions of the corresponding vectorstock.After 24 h of incubation,cells expressing fluorescent EGFPwere scored directly under a fluorescence microscope 17.2.2.Total RNA extractionTissueratsamplesfromcerebellum,cortex,hippocampus,spleen,heart,liver and kidney were collected and homogenized in TrizolReagent(Gibco-BRL,Rockville,Md,USA)inaglass-teflonpotterby10strokes,waiting 1 min and giving another 10 strokes.RNA isolationand purification was carried out following manufacturer protocols.Finally,RNA pellet was resuspended in DEPC(diethylpirocarbonate,Sigma Saint Louis,MO,USA)0.2%RNAse free water.Total RNAsamplesweretreatedwithDNAseI(AmbionEuropeLtd.,Switzerland)toeliminateanygenomicorvectorDNAcontamination.DNAse was inactivated by adding the inactivation mix(Ambion).When indicated,RNA was extracted from a hippocampus frac-tion circumscribed to the injection area to avoid dilution of trans-gene expression.2.3.DNA isolationRat hippocampus were homogenized in 10 mM TriseHCl(pH 8),0.1 M EDTA,0.5%SDS,50mg/ml proteinase K(Roche DiagnosticsGmbH,Mannheim,Germany);then,the homogenate was incu-bated 4 hours at 55?C.DNA was isolated by extraction with phe-nol:chloroform:isoamylalcohol(25:24:1v/v).Theresultantaqueous phase was extracted with chloroform;then,it wasprecipitated by centrifugation with four volumes of absoluteethanol and NaCl(final concentration 0.25 M).The pellet contain-ing the DNA was washed with ethanol 70%and resuspended inwater.2.4.RNAse protection assay(RPA)For NR1 riboprobes construction,pN60 plasmid was used astemplate 18.pN60 contains the sequence of the NR1 subunitNMDA receptor.Sense and antisense riboprobes were generated byin vitro transcription of the NheI(New England Biolabs(NEB),Ips-wich,MA,USA)and BssHII(NEB)linearized pN60 respectively,inthe presence of 60mCi of a-P32UTP(3000 Ci/mmol)(Amersham,Les Ulis,France).For RPA assay,15mg of total RNA from cerebralcortex or hippocampus were co-precipitated with 105cpm of senseorantisenseriboprobe,resuspendedinhybridizationbuffer(40 mM PIPES pH 6.7,400 mM NaCl,1 mM EDTA,80%v/v deionizedformamide)(all reactives from Sigma)and denatured at 90?C.Annealing was carried out at 45?C overnight.Then,RNaseA(MBIFermentas,Germany)was added and digestion was performedat 37?C.Reaction was stopped by addition of SDS(Sigma)andproteinase K(Roche)to a final concentration of 0.6%(w/v)and0.1 mg/ml,respectively.Samples were then extracted with phe-nol:chloroform:isoamyl alcohol(25:24:1)and precipitated withabsolute ethanol using yeast tRNA(10 mg/ml)as carrier.Denaturedsamples were analyzed by sequencing gels(6%polyacrylamide(Sigma),42%urea(Sigma).2.5.Reverse transcription(RT)1mg of total RNA was used as template in RT reaction,which wascarried out inparallel with both RevertAidTM H(-)M-MuLV ReverseTranscriptase(MBI Fermentas),and Omniscript RT Kit(QiagenHilden,Germany),following instructions provided by the manu-facturers.Briefly,total RNA was mixed with 10mM primer(oligodT,random hexanucleotides or specific primers),incubated 5 min at70?C,and kept on ice for 2 min to allow hybridization.Then,RTreaction Mix(buffer 5X,dNTPs mix 10 mM each one,RNaseinhibitor RNaseOUT(40 U/ml)(Invitrogen)and Reverse Transcrip-tase were added following manufacturer instructions.After 60 minincubation at 42?C(M-MuLV)or 40?C(Omniscript)the RTenzymewas heat inactivated.In each case the total reaction volume was30ml.2.6.Standard polymerase chain reaction(PCR)amplificationPCR was carried out with 1ml of RT product,1mM forward primerand reverse primer and Eppendorf?MasterMix(2.5?)(EppendorfAG,Hamburg,Germany).In all cases,total reaction volume was25ml.Two PCR protocols were used:either(1)2 min at 95?C,followed by 30 s at 95?C for denaturation,1 min at 60?C forhybridization and 1 min at 72?C for extension(35 cycles)or(2)15 sat 95?C for denaturation,15 s at 60?C for hybridization,30 sat 72?C for extension(35 cycles).In all cases,5ml of PCR productswere analyzed in 2%agarose gels containing ethydium bromide(both from Sigma).2.7.Real time PCR amplificationReal time PCR was carried out using 1ml of RT product,1mMforward primer and reverse primer and Quanti Tect?SYBR?GreenM.F.Adrover et al./Biochimie 92(2010)1839e18461840PCR Master Mix(Qiagen).In all cases,total reaction volume was20ml.Real time PCR assays were carried out with a LightCyclersystem(Roche Molecular Systems,California,USA).Reactions wereincubated 15 min at 95?C for the DNA polymerase HotStarTaqactivation.PCR cycle protocol was:15 s at 95?C for denaturation,15 s at 60?C for hybridization,30 s at 72?C for extension(45cycles),followed by a progressive temperature increase of 0.1?C/s,from 68?C to 95?C,finishing with 30 s at 40?C for cooling.Spec-ificity of amplification to each primer pairs was verified for thepresence of one peak in the melting curve.2.8.PrimersPrimer sequences used were obtained from the literature or byPrimer3 software 19 and are summarized in Table 1 for NR1 geneand in Table 2 for the other genes concerning the present work.Table 1 contains sequence,position and fragment size of primersused in the analysis of NR1 mRNA and genomic DNA.Table 2contains primer sequences for the other genes analyzed by RT-PCR.2.9.AnimalsAdult male Wistar rats(2e3 months old;200e250 g bodyweight)from our own animal house were used.All the proceduresinvolving animals were carried out in accordance to the guidelinesof the USA National Institutes of Health Guide for the Care and Useof Laboratory Animals(1996).Protocols were approved by theAnimal Care and Use Committees from the University of BuenosAires,Argentina(2004).3.Results3.1.Detection of NR1 antisense RNAWith the aim to find out if exogenous NR1 antisense RNA wasexpressed from an amplicon vector,three groups of rats wereinjected into the hippocampus with vectors expressing EGFP mRNAand one of the following sequences:1)NR1 mRNA(NR1 vector,S),2)NR1 antisense(NR1 antisense vector,AS)or 3)LacZ mRNA(Z)20.There was a fourth group which consisted in vehicle-injectedrats(Vh).RT-PCR assays were carried out using total hippocampalRNA samples from these rats.Reverse transcription reaction wasperformed using a forward(50)specific primer for NR1 sequence.Inthe subsequent PCR the cDNA was amplified by using specificprimers for NR1.In all four samples,coming from animals eitherinjected or not with NR1 antisense vector,we detected a band thatcould be interpreted as coming from an NR1 antisense(Fig.1a),suggesting that this amplification could be originated from anendogenously expressed RNA molecule.Since the bands wererather similar in every case(Fig.1a),RNA was then extractedexclusively from the hippocampus fraction circumscribed to theinjection area.The corresponding RT-PCR assay showed that theexpression of the putative NR1 antisense was higher in samplescoming from NR1 antisense vector injected animals,althougha band appeared in all cases(Fig.1b).To further investigate thenature of this product,which appeared even when the transgenewas notinjected,wedecided to perform an RT-PCR assay using RNAextracted from different tissues.Total RNAs from liver,spleen,heart,kidney,cerebral cortex and hippocampus from naive non-injected rats were used(Fig.1c).A band similar to that reportedabove,matching with NR1 antisense,was exclusively present insamples coming from nervous tissue,i.e.cerebral cortex andhippocampus,where NR1 mRNA is endogenously and conspicu-ously expressed.This result raised the possibility that the amplifiedfragment could be originated from the NR1 mRNA.However,at thispoint wewere notable to discard that the amplified fragment couldhave beenoriginatedfrom anothertranscript coming froma hypothetic genomic antisense sequence.Therefore,to elucidate ifthe amplified fragment was originated from an endogenous anti-sense or from a mRNA,three different approaches were followed:sequencing of PCR products,RT-PCR assay under different condi-tions and RNAse protection assay(RPA).The first step was to sequence the products of PCR after RT fromtotal hippocampal RNA samples performed either with reverse(30)or forward(50)primers.The corresponding sequences from bothreactions resulted identical(data not