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Modification of a commercial DNA extraction kit for
safe and rapid recovery of DNA and RNA
simultaneously from soil, without the use of harmful
solvents
Estelle Tournier, Laurie Amenc, Anne-Laure Pablo, Elvira Legname-Vonarx,
Eric Blanchart, Claude Plassard, Agnès Robin, Laetitia Bernard
To cite this version:
Estelle Tournier, Laurie Amenc, Anne-Laure Pablo, Elvira Legname-Vonarx, Eric Blanchart, et al..
Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA
simultaneously from soil, without the use of harmful solvents. MethodsX, Elsevier, 2015, 2,
pp.182-191. �10.1016/j.mex.2015.03.007�. �hal-01268966�
Modi
fication
of
a
commercial
DNA
extraction
kit
for
safe
and
rapid
recovery
of
DNA
and
RNA
simultaneously
from
soil,
without
the
use
of
harmful
solvents
E.
Tournier
a,1,2,
L.
Amenc
b,2,
A.L.
Pablo
a,2,
E.
Legname
b,
E.
Blanchart
d,
C.
Plassard
b,
A.
Robin
c,
L.
Bernard
d,*
a
IRD,UMREco&Sols,2PlaceViala,34060 MontpellierCedex1,France
b
INRA,UMREco&Sols,2PlaceViala,34060MontpellierCedex1,France
c
CIRAD,UMREco&Sols,2PlaceViala,34060MontpellierCedex1,France
d
IRD,UMREco&Sols-LaboratoiredesRadioIsotopes(LRI),Ampandrianomby,Antananarivo101,Madagascar GRAPHICAL ABSTRACT
ABSTRACT
Anoptimizedmethod,basedonthecouplingoftwocommercialkits,isdescribedfortheextractionofsoilnucleic acids,withsimultaneousextractionandpurificationofDNAandRNAfollowingacascadeschemeandavoiding theuseofharmfulsolvents.TheprotocolcanmonitorthevariationsintherecoveryyieldofDNAandRNAfrom soilsofvarioustypes.Thequantitativeversionoftheprotocolwasobtainedbytestingthestartingsoilquantity, thegrindingparametersandthefinalelutionvolumes,inordertoavoidsaturationofbothkits.
Afirstsoil-crushingstepinliquidnitrogencouldbeaddedfortheassessmentoffungalparameters.
*Correspondingauthor.
E-mailaddress:Laetitia.Bernard@ird.fr(L. Bernard).
1
Presentaddress:CIRAD,UMRLSTM,CampusdeBaillarguet,34398MontpellierCedex5,France.
2
Theseauthorscontributedequallytothiswork.
http://dx.doi.org/10.1016/j.mex.2015.03.007
2215-0161/ã2015PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/ licenses/by/4.0/).
ContentslistsavailableatScienceDirect
MethodsX
Theprotocolwasefficientondifferenttropicalsoils,includingAndosol,whiletheirhighcontentsofclays, includingpoorlycrystallineclays,andFeandAloxidesusuallymakethenucleicacidextractionmoredifficult. TheRNArecoveryyieldfromtheprevioustropicalsoilsappearedtocorrelatebettertosoilrespirationthan
DNA,whichispositivelyinfluencedbysoilclaycontent.
ã2015PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons. org/licenses/by/4.0/). ARTICLE INFO
Methodname:SoilDNA/RNAco-extraction
Keywords:SoilDNAandRNA,Quantitativeco-extraction,Molecularbiomass,Tropicalsoil
Articlehistory:Received8January2015;Accepted26March2015
1.Methoddetails
WeadaptedacommercialDNAextractionkit(FastDNASPINTMkitforsoil,MPBiomedical,Santa Anna,CA),torecoverDNAandRNAsimultaneously,avoidingtheuseofhazardoussolvents.ThisDNA kit,basedonanSDSextractionbuffer,hasalreadyshownitsefficiency[1,2].RNArecoverywasallowed becausespecialcaresweretakentoworkunderRNase-freeconditions.Allsolutionsandglassware weretreatedwithdiethylpyrocarbonate(DEPC)toensurethattheywereRNasefreeandonlycertified RNase-andDNase-freeplastictubeswereused.Eachcentrifugationstepwasperformedat4Cand tubeswereplacedonicewhilewaitingforthenextstepoftheprocedure.RNAcouldthereforebe purifiedfromtheDNAwashingsolutionatthestep8oftheprotocolasshownontheflowchart presentedinFig.1andasfurtherdescribed.Modificationswebroughttotheinitialprotocolofthe manufactureraredetailedinitaliccharacter.
1.Soilsamplepreparation:add250mgofsoilsample(characteristicsofsoilsusedinthisstudyare showninTable1)toaLysingMatrixEtubeandfreezeovernightat80C.Initiallythemanufacturer
preconizedtouse500mgofsoil.Wehavetested125,250,500and750mg,andobservedthat500mgof soilsamplesledalreadytothemaximumofDNArecovered,as750mgofsoildidnotimprovetheyield (Fig.2).Thereforeevenif500mgofsoilincreasedtheRNAyieldbyafactorofalmost4,thesoilsamples beingprocessedseemedtobelimitedto250mgtokeeptheextractedDNAbelowthesaturationlimitof thekitandtobeabletofollowvariationsofbothmolecules.Ofcourse500mghastobechosenifthe objectiveisonlytomaximizethequantityofDNAandRNArecoveredandnottorelatetheirrecovery yieldtomolecularbiomassestimation.
2.Cellslysing:add978
m
Lsodiumphosphatebuffer,122m
LofMTbuffer.Whenextractingnucleicacids fromandosol,20mgcaseinehastobeaddedtothelysingmixinordertosaturatethehighadsorption capacityofthenon-crystallizedclays[3].3.Grinding:homogenizeintheFastPrepTMinstrument(MPBiomedical)for40sataspeedsettingof
6.0.Coolsampleonicefor5minandperformanothergrindingstepundersimilarconditions(40s, speed6.0).Initiallythemanufacturerpreconizedasinglehomogenizationstep.Toincreasetheamount ofnucleicacidsrecoveredforfurtheranalyses,asecondgrindingstepwasintroduced(Fig.3).
4.Centrifugeat14,000gfor5minat4C.Transfersupernatanttoaclean2.0mLmicrocentrifuge tube.
5.Proteinprecipitation:add250
m
LPPS(proteinprecipitationsolution)andmixbyshakingthetube byhand10times.Keeponicebeforenextstep,whenprocessingothersamples.6.Centrifugeat14,000gfor5minat4Ctopelletprecipitate.Transfersupernatanttoaclean2mL microcentrifugetube.
7.DNAbinding:re-suspendBindingMatrixsuspensionandadd1.0mLtosupernatantinthe2mL tube.Placeonrotaryshakerfor12min,23rpmatroomtemperature, toallowbindingofDNA. Initiallythemanufacturerpreconizeda5minbindingtimebutwehaveobservedthatincreasingthe DNAbindingtimefrom5to12minavoidedDNAcontaminationintothewashingsolutioncontaining RNA.ThiswasverifiedbyPCR(datanotshown).
8.Centrifugeat14,000gfor2minat4CtopelletDNAbindingmatrix.Removethesupernatantand transferitintoaclean15mLtubeforfurtherRNApurification.Keeponicebeforeprocessing.
a.DNApurificationprocedure:
9a. Re-suspendtheDNABindingMatrixin500
m
Lofguanidinethiocyanate(5.5M)andtransferto aSPINTMFiltercolumnandcentrifugeat14,000gat4Cfor1min.TransfertheeluatefromthecatchtubetotheRNA15mLtube(seestep8).
10a.Add500
m
LpreparedSEWS-M(ethanoladded)andgentlyre-suspendthepelletusingtheforce oftheliquidfromthepipettip.11a. Centrifugeat14,000gat4Cfor1min.Emptythecatchtubeandreplace.
12a. Withoutanyadditionofliquid,centrifugeasecondtimeat14,000gfor2minutesto“dry”the matrixofresidualwashsolution.Discardthecatchtubeandreplacewithanew,cleancatch tube.
13a.AirdrytheSPINTMFilterfor5minatroomtemperature.
Fig.2.Concentrationsofnucleicacidsextracted(DNAandRNA)infunctionofthequantityofsoilsubmittedtotheextraction procedure.
Table1
CharacterizationofthesoilsusedtodevelopandtotesttheDNA/RNAco-extractionmethod.
Soil Maugio Andranomanelatra Betafo Lazaina Miandrivazo GPSordination 35900300E, 43701400N 1946042.1900S, 4706028.7000E 1950005.9200S, 4650035.4900E 1846054.4500S, 4732005.9900E 1932051.1500S, 4528008.4300E WaterpH 8.29 4.42 6.25 6.14 6.55 Clay,g(kgsoil)1 248 619 149 340 90 Silt,g(kgsoil)1 436 188 469 70 194 Sand,g(kgsoil)1 350 193 382 580 716 AlDCBa,g(kgsoil)1 ND 19.9 28.7 7.2 1.44 FeDCBa,g(kgsoil)1 ND 62.1 60.3 35.13 8.61 SiDCBa,g(kgsoil)1 ND 4.2 1.3 0.98 4.15 Aloxb,g(kgsoil)1 ND 17.8 34.6 2.95 1.48 Feoxb,g(kgsoil)1 ND 4.5 24.3 1.41 1.77 Sioxb,g(kgsoil)1 ND 1.9 15.9 0.24 0.73 Alp ND 5.72 6.78 3.04 0.29 Fep ND 3.04 2.06 1.73 0.23 Sip ND <0.01 0.34 0.72 0.73 Carbon,g(kgsoil)1 9.30 60.24 25.00 20.34 11.73 C/N 10.40 12.73 13.15 14.95 11.61 CECcmol(kgsoil)1 21.10 17.32 29.59 6.20 14.25 ND:notdetermined.
14a.Gentlyre-suspendBindingMatrix(abovetheSPINfilter)in150
m
LofDES(DNase/pyrogen-free water)andincubateat55Cfor12min.Thefinalelutionvolumewasincreasedcomparedto theinitialprotocol,from100to150m
LtoincreaseDNArecoveryfromthematrix(Fig.4).15a.Centrifugeat14,000gfor1mintobringelutedDNAintothecleancatchtube.Discardthe SPINfilter.DNAisnowreadyfordownstreamapplication.Hereendedthemodifiedprotocolof theFastDNASPINTMkitforsoil.
b.RNApurificationprocedure:
9b.Add1volumeofisopropanoland500
m
Lofsodiumacetate(3MpH4)tosupernatantobtainedat step8and9a(usually2.5mL).Gentlyshakethetubeandincubatefor90minat20C.10b. Centrifugeat14,000gand4Cfor30minandwashthepelletwith70%ethanolandcentrifuged again(14,000gat4Cfor5min).Removeethanolandair-drythepelletfor5min.
11b.Re-suspendthepelletin100
m
LofRNAase-freewaterandkeepinicefor10min.12b.Add300
m
Lofsaltsolution(RNaidkit-MPBiomedicals)andtransferintoanew1.5mLtube.13b. Add5
m
LofRNABindingMatrixandgentlyshake15minatambienttemperature.14b. Centrifugeat14,000gand4Cfor1min.Discardthesupernatantandre-suspendthepellet
in500
m
LofRNAwashsolution.15b. Centrifugeat14,000gand4Cfor1minremovethesupernatant.Re-suspendthepelletin 60
m
LofRNasefreewater,gentlyshakeandincubateat55Cfor15min.Thefinalelutionvolume wasincreasedcomparedtothe initialprotocolfrom20to60m
L(Fig.5).Witha60m
LelutionFig.3. Concentrationsofnucleicacidsextracted(DNAandRNA)infunctionofthenumberof40-sgrindingcyclesat6ms1 performedbytheFastPrepTMinstrument.Errorsbarscorrespondto95%confidenceintervals(alpha0.05,3replicates).The
conditionschosenasstandardareframed.
volume,RNAyieldcouldstillbeimproved.However,increasingtheelutionvolumewouldleadtoa dilutionandhenceaconcentrationtoolowforsubsequentreversetranscriptasereactions.Transfer thesupernatanttoaclean1.5mLRNAasefreetubeandkeepat80Cpriortofurtherapplication.
2.Nucleicacidsquantitation
TheDNAand RNAwerequantifiedbyfluorometry usingtheQuant-iTTMPicoGreenDNA and
Quant-iTTM RiboGreen RNA assaykit (Molecular Probes, Carlsbad, New Mexico) respectively in
accordancewiththemanufacturer’sinstructions.ThepurityandintegrityoftheRNArecoveredwas alsoverifiedaftermigrationona1%agarosegel(Fig.6).
3.Optionforfungalparametersanalyses
For the analyses of fungal parameters (density and diversity), a sample size of 1g hasbeen recommendedinordertoberepresentativeof thecommunity[4].Therefore, theapplicabilityto
Fig.5.QuantityofRNArecoveredasafunctionofthevolumeofelutionsolution.Theconditionschosenasstandardareframed.
Fig.6.Electrophoreticprofiles(agarose1%)of10mLofthefinalRNAextract(step15boftheprotocol),and12mLofthe1kb ladder(Invitrogen,France).Gelwasstainedwithethidiumbromide.
fungalbiomassquantification(seemethodbelow)wastestedbycrushingandhomogenizing30gof soilinliquidnitrogeneitherbyhand,orusingamortargrinder(Pulverisette2,Fritsch,Idar-Oberstein, Germany)priortosubsample0.25gandthenbeginningtheprotocol.Fromourresults,itappearsthat crushing 30g of soil in liquid nitrogen prior to subsample 0.25g for the further co-extraction significantlyimprovedtherecoveryof18SgenescopiesfromtheDNAwhileithasnoeffectonthe 16Scopynumber(Fig.7).ThequantitativePCRprotocolusedisdescribedinSupplementarymaterial (S1).
Fig.7.(a)Bacterial(16S)and(b)fungal(18S)ribosomalgenecopynumberspergramofsoil,measuredbyqPCRasafunctionof thesoilhomogenizationtreatmentbeforesubsampling(0.25gand0.50gofsoil)fortheco-extractionprotocol.30gofsoilwere usedperhomogenizationtreatment.Errorsbarscorrespondto95%confidenceintervals(alpha0.05,3replicates).
4.Analysesofsamples
Theco-extractionprotocolwasoptimizedonasandyclayloamsoilfromSouthofFrance(Maugio– Table1).Yieldsof26.7
m
gofpurifiedDNAand4.5m
gofpurifiedRNApergramofsoilwereobtained, withaverygoodrepeatabilityforbothDNAandRNA(coefficientsofvariationof8.69%forDNAand 5.25%forRNAwhenappliedtoasetof10replicatedsoilsamples).Theprotocolwastestedon4tropical soils from Madagascar, which were chosen because their composition was thought likely to complicatetheextractionofnucleicacids(Table1).Onemajorproblemisthepresenceofminerals knowntoadsorborganicmoleculesstrongly,namelyclays(Andranomanelatra,BetafoandLazaina) andmetaloxides(allsoils),andespeciallywhenclaysarepoorlycrystallized(Betafoandtoalesser extendAndranomanelatra).Anotherproblemisthesmallsizeofthemicrobialbiomasspool,whichis usuallycharacteristicofcarbon depletedsandysoil(Miandrivazo).Themostdifficultsoilwasthe Andosol containing allophanes (Betafo), which required the addition of 20mg caseine to the extractionmixjustbeforetheFastPrepgrindingstep(protocolstep2).Thefoursoilswereextractable andgavevariousDNAandRNArecoveryyieldssufficientlyconcentratedtobefurtheranalyzedbyany othermoleculartechnique(Fig.8).Asexpected,themeanRNA/DNAratioobservedfortropicalsoils (0.62)wasgreaterthanthatfortheMediterraneansoilsampledduringthewinter-time(0.17). 5.AdditionalinformationVariationsintheDNAandRNAcompositionbringadditionalinformationasDNAislinkedtocell replicationwhileRNAislinkedtoproteinsynthesis.Acomparisonofthecompositionofmicrobial communitiesbasedonco-extractionofbothDNAandRNAcanprovideaninsightintotheecologyof populations,providedthatDNAandRNAaresubjectedtothesameextractionbias.However,todate, veryfewstudiesdevelopedmethodsforDNA/RNAco-extractionfromsoilsamples[5–10].Withthe exceptionofthemethoddescribed by[8],allprocedureshaverequiredharmfulsolventssuchas
Fig.8.DNA(black)andRNA(lightgrey)extractionyieldsandtheirrespective95%confidenceintervals(alpha0.05,3replicates) from4tropicalsoils,sampledinMadagascarandcharacterizedbydifferenttextures,mineralogies,metalandcarboncontents. SoiltypesareindicatedfollowingtheWRBnomenclature,andfullcharacterizationispresentedinTable1.
phenol,chloroformand
b
-mercaptoethanol,whicharecarcinogenic,mutagenicreproductivetoxins. The DNA/RNA co-extraction protocol developed in the present work used DEPC and guanidine thiocyanate,whicharejustirritantsbycontactoringestion.SDS-basedmethodsarethoughttobeless efficientthansolvent-basedmethods.Usingasolvent-basedmethod,[5]obtainedyieldsbetween10 and20m
gofDNAand2and5m
gRNApergramofsoil,inanunpurifiedextract.Inordertopurifysuch extracts,thesamplewouldhavetobedividedintotwoaliquotsandeachonedigestedwitheither DNAseorRNAsethusdecreasingtheyieldofbothRNAandDNA.Recently, the quantity of DNA extractedfrom soil had been defined as “microbial molecular biomass”andproposedasamicrobialindicatorofsoilstatus[11–14].Thisnotionofconsideringtotal recoveredDNAasatotalbiomassquantificationtoolisstillstronglydebatedinmicrobialecology becauseDNAcanremainstableintheenvironmentforlongperiodsoftime.IntheirDNAextraction protocol,[14]addedafirststepofsoilwashingtofirstdesorbtheextracellularDNAfromthemineral matrix[15].Suchstepcanalsobeaddedintothepresentprotocol.Buttheparticularityofthepresent methodistosimultaneouslyco-extractRNA,whichisamuchmorelabilemoleculethanDNA.Onthe fourprecedentsoilsfromMadagascar,wehavemeasuredtheCO2evolution(soilrespiration)atpF
2.5during6hat25C.WhenplottedtherespirationagainstDNAandRNArecoveryyieldswefounda muchbettercorrelationwithRNA(Fig.9a;R2:0.97)thanwithDNA(datanotshown;R2:0.66).This canbe explainedby thehigh affinity of extracellular DNA for clays asRNA/DNA ratioinversely
Fig.9. XYdotplotsof(a)soilrespirationagainstRNAextractionyieldsandtheirrespective95%confidenceintervals(alpha0.05, 3replicates),and(b)RNA/DNAagainstsoilclaycontent.Bestfittedrelationshipsfollowedsecondorderpolynomialfunctions. RegressioncoefficientsR2
correlatedtoclaycontent(Fig.9b).ThereforeRNArecoveryyieldshouldbeabetterindicatorofliving microbialbiomassthanDNA.Nowthisprotocolhasbeendevelopedandtested,futurestudieswillbe conducted todetermine whether theRNA/DNA ratio of themicrobial communities is a reliable bioindicatorofsoilstatusinacontextofglobalchanges(climate,landuses,agriculturalpractices...), providedthatsucheffectsarestudiedonthesamesoilorsoilswithsimilarclaycontent.
Future technical optimizations couldinclude therecoveryof messenger RNAto quantifythe expressionoffunctional genes,compared totheirpresence intotheDNAfraction, aswellasthe recoveryofenzymesfromtheprecipitateobtainedatstep5aswealreadyverifiedthatPPSdoesnot inactivatethem(datanotshown).
Acknowledgments
This study was partly funded by the French “AgenceNationale pourla Recherche” (Systerra program,PerfComANR-08-STRA-11andPépites,ANR-08-STRA-10-04projects).WethankSiobhan Stauntonfor editingtheEnglish language.The authorswould liketodedicatethis articletothe memoryoftheirhighlyrespectedandmuchlovedcolleague,ElvireLegname(INRA,UMREco&Sols). MethodsXthanksthereviewersofthisarticlefortakingthetimetoprovidevaluablefeedback. AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,athttp://dx.doi. org/10.1016/j.mex.2015.03.007.
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