Removal of Contaminating Genomic DNA in QRT-PCR Using a … · 2009. 10. 27. · Thermo...
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Conclusion • The current data suggest that shrimp nuclease has no inhibitory effect on 1-step or 2-step QRT- PCR and the integrity of RNA and cDNA is not altered. • Shrimp nuclease effectively removes any contaminating DNA without adding an extra step to the QRT-PCR protocol; thus, completely eliminating the need for harsh and time-consuming DNase I treatment. In doing so, shrimp nuclease increases the accuracy and reproducibility of QRT-PCR reactions, especially when using crudely purified samples. • Shrimp nuclease can also be used for removal of amplicon carry-over contaminants in the PCR reaction. † For more information contact ian.kavanagh@thermofisher.com Introduction DNA contamination can often occur in quantitative reverse transcription – polymerase chain reactions (QRT-PCR), and should be removed in order to avoid false positive results. DNase I is commonly used for removing DNA contamination, but this has a relatively long and harsh protocol which introduces an extra step between the isolation of RNA and the QRT-PCR reaction itself, as well as increasing the risk of RNA degradation due to the harsh inactivation conditions. An endonuclease from arctic shrimp Pandalus borealis has properties that make it useful for the removal of contaminating DNA. The endonuclease activity of the enzyme is specific to double stranded DNA, which therefore allows the enzyme to be added directly into the reverse transcription step. Unlike DNase I, the shrimp nuclease is easily inactivated at high temperatures, such as those used for the RT deactivation / hot start incubation step of a QRT-PCR reaction, and therefore can be used to selectively degrade double stranded DNA, leaving single stranded DNA and RNA intact. Aim To determine whether addition of shrimp nuclease can remove the contaminating DNA from the QRT-PCR reaction without having any inhibitory effects. Method To evaluate whetherThermo Scientific shrimp nuclease (AB-1311) would inhibit QRT-PCR reaction, 0.1, 0.2 and 0.4 units of the enzyme were added / reaction, in conjunction with other reagents from the Thermo Scientific Verso ™ QRT-PCR Kit (AB-4100). Five 10-fold dilutions (100 ng, 10 ng, 1 ng, 100 pg and 10 pg) of Human LiverTotal RNA (Ambion Europe Ltd, Huntingdon) were used as template to amplify 74 bp fragment of Apolipoprotein-B gene using a Stratagene Mx3005P QPCR instrument (Stratagene, Leicester). To test the ability of shrimp nuclease to effectively eliminate contaminating DNA, 0.4 units of shrimp nuclease were pre-incubated with four 10-fold dilutions (100, 10, 1 and 0.1 ng) of human genomic DNA (Sigma-Aldrich, Dorset) at 50°C for 30 min prior to the QPCR reaction. Amplification of apolipoprotein-B gene (74 bp) was carried out on Stratagene Mx3005P QPCR instrument using Thermo Scientific ABsolute ™ Blue QPCR Probe Mix (AB-4132). As a control, all template dilutions were incubated with shrimp nuclease buffer without the enzyme.The percentage removal of genomic DNA was calculated by comparing the ∆C T values between reactions where DNA was pre-incubated with shrimp nuclease to reactions incubated with buffer. All experiments were performed three times with triplicate reactions. Results Figure 1 shows that addition of shrimp nuclease (0.1 to 0.4 units) in the QRT-PCR reaction mix had no adverse effects on the integrity of RNA. In addition the result confirmed that amplification of Apolipoprotein B gene across five 10-fold dilutions of RNA was not inhibited by any of the three concentrations of shrimp nuclease tested for these experiments (Figure 1). Table 1 and Figure 2 show that pre-QPCR incubation of 100, 10, 1 and 0.1 ng of human genomic DNA with 0.4 units of shrimp nuclease caused retardation of C T values in test samples compared to the controls (buffer only without the shrimp nuclease). A late C T value is indicative of low quantities of the starting template. A notable 99-100% removal of DNA following incubation with 0.4 units of shrimp nuclease validates efficacy of this enzyme to eliminate contaminating DNA from QRT-PCR reactions (Table 1 and Figure 2). Removal of Contaminating Genomic DNA in QRT-PCR Using a Shrimp Nuclease Nicky Quispe, Saima Naveed Nayab and Ian Kavanagh † Thermo Fisher Scientific,ABgene House, Blenheim Road, Epsom, Surrey, KT19 9AP, United Kingdom. Figure 2: Human genomic DNA (100 - 0.1 ng) was pre-incubated with 0.4 units / reaction of shrimp nuclease (red curves) and with the buffer only (black curves) before amplification of a 74 bp fragment of theApolipoprotein B gene on Stratagene Mx3005P. Figure 1: 1-step QRT-PCR results showing amplification of Apolipoprotein-B gene using 100 ng -10 pg of input Human LiverTotal RNA following incubation with 0.1 (green), 0.2 (blue), 0.4 (red) units of shrimp nuclease per reaction. Black curves represent water as a control used instead of the shrimp nuclease. DNA (ng) C T control (buffer only) C T test (Shrimp Nuclease) ΔC T Calculated remaining concentration of DNA after incubation with shrimp nuclease (ng) % Removal 100 21.89 29.93 -8.04 0.365 99.64 10 25.12 32.69 -7.57 0.055 99.45 1 28.27 37.17 -8.9 0.003 99.75 0.1 31.54 No C T - - 100 NTC No C T No C T - - n/a Table 1: Removal of DNA following incubation with shrimp nuclease
Transcript of Removal of Contaminating Genomic DNA in QRT-PCR Using a … · 2009. 10. 27. · Thermo...
Conclusion• Thecurrentdatasuggestthatshrimpnucleasehasnoinhibitoryeffecton1-stepor2-stepQRT-
PCRandtheintegrityofRNAandcDNAisnotaltered.
• ShrimpnucleaseeffectivelyremovesanycontaminatingDNAwithoutaddinganextrasteptotheQRT-PCRprotocol;thus,completelyeliminatingtheneedforharshandtime-consumingDNaseItreatment.Indoingso,shrimpnucleaseincreasestheaccuracyandreproducibilityofQRT-PCRreactions,especiallywhenusingcrudelypurifiedsamples.
• Shrimpnucleasecanalsobeusedforremovalofampliconcarry-overcontaminantsinthePCRreaction.
† For more information contact [email protected]
IntroductionDNAcontaminationcanoftenoccurinquantitativereversetranscription–polymerasechainreactions(QRT-PCR),andshouldberemovedinordertoavoidfalsepositiveresults.DNaseIiscommonlyusedforremovingDNAcontamination,butthishasarelativelylongandharshprotocolwhichintroducesanextrastepbetweentheisolationofRNAandtheQRT-PCRreactionitself,aswellasincreasingtheriskofRNAdegradationduetotheharshinactivationconditions.
AnendonucleasefromarcticshrimpPandalus borealishaspropertiesthatmakeitusefulfortheremovalofcontaminatingDNA.TheendonucleaseactivityoftheenzymeisspecifictodoublestrandedDNA,whichthereforeallowstheenzymetobeaddeddirectlyintothereversetranscriptionstep.UnlikeDNaseI,theshrimpnucleaseiseasilyinactivatedathightemperatures,suchasthoseusedfortheRTdeactivation/hotstartincubationstepofaQRT-PCRreaction,andthereforecanbeusedtoselectivelydegradedoublestrandedDNA,leavingsinglestrandedDNAandRNAintact.
AimTodeterminewhetheradditionofshrimpnucleasecanremovethecontaminatingDNAfromtheQRT-PCRreactionwithouthavinganyinhibitoryeffects.
MethodToevaluatewhetherThermoScientificshrimpnuclease(AB-1311)wouldinhibitQRT-PCRreaction,0.1,0.2and0.4unitsoftheenzymewereadded/reaction,inconjunctionwithotherreagentsfromtheThermoScientificVerso™ QRT-PCR Kit(AB-4100).Five10-folddilutions(100ng,10ng,1ng,100pgand10pg)ofHumanLiverTotalRNA(AmbionEuropeLtd,Huntingdon)wereusedastemplatetoamplify74bpfragmentofApolipoprotein-BgeneusingaStratageneMx3005PQPCRinstrument(Stratagene,Leicester).
TotesttheabilityofshrimpnucleasetoeffectivelyeliminatecontaminatingDNA,0.4unitsofshrimpnucleasewerepre-incubatedwithfour10-folddilutions(100,10,1and0.1ng)ofhumangenomicDNA(Sigma-Aldrich,Dorset)at50°Cfor30minpriortotheQPCRreaction.Amplificationofapolipoprotein-Bgene(74bp)wascarriedoutonStratageneMx3005PQPCRinstrumentusingThermoScientificABsolute™BlueQPCRProbeMix(AB-4132).Asacontrol,alltemplatedilutionswereincubatedwithshrimpnucleasebufferwithouttheenzyme.ThepercentageremovalofgenomicDNAwascalculatedbycomparingthe∆CTvaluesbetweenreactionswhereDNAwaspre-incubatedwithshrimpnucleasetoreactionsincubatedwithbuffer.Allexperimentswereperformedthreetimeswithtriplicatereactions.
ResultsFigure1showsthatadditionofshrimpnuclease(0.1to0.4units)intheQRT-PCRreactionmixhadnoadverseeffectsontheintegrityofRNA.InadditiontheresultconfirmedthatamplificationofApolipoproteinBgeneacrossfive10-folddilutionsofRNAwasnotinhibitedbyanyofthethreeconcentrationsofshrimpnucleasetestedfortheseexperiments(Figure1).Table1andFigure2showthatpre-QPCRincubationof100,10,1and0.1ngofhumangenomicDNAwith0.4unitsofshrimpnucleasecausedretardationofCTvaluesintestsamplescomparedtothecontrols(bufferonlywithouttheshrimpnuclease).AlateCTvalueisindicativeoflowquantitiesofthestartingtemplate.Anotable99-100%removalofDNAfollowingincubationwith0.4unitsofshrimpnucleasevalidatesefficacyofthisenzymetoeliminatecontaminatingDNAfromQRT-PCRreactions(Table1andFigure2).
Removal of Contaminating Genomic DNA in QRT-PCR Using a Shrimp Nuclease
NickyQuispe,SaimaNaveedNayabandIanKavanagh†
ThermoFisherScientific,ABgeneHouse,BlenheimRoad,Epsom,Surrey,KT199AP,UnitedKingdom.
Figure 2:HumangenomicDNA(100-0.1ng)waspre-incubatedwith0.4units/reactionofshrimpnuclease(redcurves)andwiththebufferonly(blackcurves)beforeamplificationofa74bpfragmentoftheApolipoproteinBgeneonStratageneMx3005P.Figure 1:1-stepQRT-PCR
resultsshowingamplificationofApolipoprotein-Bgeneusing100ng-10pgofinputHumanLiverTotalRNAfollowingincubationwith0.1(green),0.2(blue),0.4(red)unitsofshrimpnucleaseperreaction.Blackcurvesrepresentwaterasacontrolusedinsteadoftheshrimpnuclease.
DNA (ng) CT control(buffer only)
CT test(Shrimp Nuclease)
ΔCT Calculated remaining concentration of DNA after incubation with shrimp nuclease (ng)
% Removal
100 21.89 29.93 -8.04 0.365 99.64
10 25.12 32.69 -7.57 0.055 99.45
1 28.27 37.17 -8.9 0.003 99.75
0.1 31.54 NoCT - - 100
NTC NoCT NoCT - - n/a
Table 1:RemovalofDNAfollowingincubationwithshrimpnuclease
![QRT Report [2001-2005]](https://static.fdocuments.net/doc/165x107/588c6afc1a28abbe218b82c4/qrt-report-2001-2005.jpg)
