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

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:[email protected](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

WeadaptedacommercialDNAextractionkit(FastDNASPINTMkitforsoil,MPBiomedical,Santa Anna,CA),torecoverDNAandRNAsimultaneously,avoidingtheuseofhazardoussolvents.ThisDNA kit,basedonanSDSextractionbuffer,hasalreadyshownitsefficiency[1,2].RNArecoverywasallowed becausespecialcaresweretakentoworkunderRNase-freeconditions.Allsolutionsandglassware weretreatedwithdiethylpyrocarbonate(DEPC)toensurethattheywereRNasefreeandonlycerti_fied RNase-andDNase-freeplastictubeswereused.Eachcentrifugationstepwasperformedat4Cand tubeswereplacedonicewhilewaitingforthenextstepoftheprocedure.RNAcouldthereforebe puri_fiedfromtheDNAwashingsolutionatthestep8oftheprotocolasshownonthe_flowchart presentedinFig.1andasfurtherdescribed.Modificationswebroughttotheinitialprotocolofthe manufactureraredetailedinitaliccharacter.

1.Soilsamplepreparation:add250mgofsoilsample(characteristicsofsoilsusedinthisstudyare showninTable1)toaLysingMatrixEtubeandfreezeovernightat80_{C.Initiallythemanufacturer} preconizedtouse500mgofsoil.Wehavetested125,250,500and750mg,andobservedthat500mgof soilsamplesledalreadytothemaximumofDNArecovered,as750mgofsoildidnotimprovetheyield (Fig.2).Thereforeevenif500mgofsoilincreasedtheRNAyieldbyafactorofalmost4,thesoilsamples beingprocessedseemedtobelimitedto250mgtokeeptheextractedDNAbelowthesaturationlimitof thekitandtobeabletofollowvariationsofbothmolecules.Ofcourse500mghastobechosenifthe objectiveisonlytomaximizethequantityofDNAandRNArecoveredandnottorelatetheirrecovery yieldtomolecularbiomassestimation.

2.Cellslysing:add978

m

Lsodiumphosphatebuffer,122

m

LofMTbuffer.Whenextractingnucleicacids fromandosol,20mgcaseinehastobeaddedtothelysingmixinordertosaturatethehighadsorption capacityofthenon-crystallizedclays[3].

3.Grinding:homogenizeintheFastPrepTM_{instrument(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.DNApuri_ficationprocedure:

9a. Re-suspendtheDNABindingMatrixin500

m

Lofguanidinethiocyanate(5.5M)andtransferto aSPINTM_{Filtercolumnandcentrifugeat14,000gat4}_{Cfor1min.Transfertheeluatefromthe} catchtubetotheRNA15mLtube(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.AirdrytheSPINTM_{Filterfor5minatroomtemperature.}

Fig.2.Concentrationsofnucleicacidsextracted(DNAandRNA)infunctionofthequantityofsoilsubmittedtotheextraction procedure. 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,from100to150

m

LtoincreaseDNArecoveryfromthematrix(Fig.4). 15a.Centrifugeat14,000gfor1mintobringelutedDNAintothecleancatchtube.Discardthe

SPINfilter.DNAisnowreadyfordownstreamapplication.Hereendedthemodifiedprotocolof theFastDNASPINTM_kitforsoil.

b.RNApurificationprocedure:

9b.Add1volumeofisopropanoland500

m

Lofsodiumacetate(3MpH4)tosupernatantobtainedat step8and9a(usually2.5mL).Gentlyshakethetubeandincubatefor90minat20_C.

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. Centrifugeat14,000_gand4_{Cfor1min.Discardthesupernatantandre-suspendthepellet} in500

m

LofRNAwashsolution.

15b. Centrifugeat14,000gand4Cfor1minremovethesupernatant.Re-suspendthepelletin 60

m

LofRNasefreewater,gentlyshakeandincubateat55Cfor15min.Thefinalelutionvolume wasincreasedcomparedtothe initialprotocolfrom20to60

m

L(Fig.5).Witha60

m

Lelution

Fig.3. Concentrationsofnucleicacidsextracted(DNAandRNA)infunctionofthenumberof40-sgrindingcyclesat6ms1 performedbytheFastPrepTM_{instrument.Errorsbarscorrespondto95%confidenceintervals(alpha0.05,3replicates).The}

conditionschosenasstandardareframed.

volume,RNAyieldcouldstillbeimproved.However,increasingtheelutionvolumewouldleadtoa dilutionandhenceaconcentrationtoolowforsubsequentreversetranscriptasereactions.Transfer thesupernatanttoaclean1.5mLRNAasefreetubeandkeepat80_{Cpriortofurtherapplication.}

2.Nucleicacidsquantitation

TheDNAand RNAwerequantifiedbyfluorometry usingtheQuant-iTTM_{PicoGreenDNA 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 signi_ficantlyimprovedtherecoveryof18SgenescopiesfromtheDNAwhileithasnoeffectonthe 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).

extractionmixjustbeforetheFastPrepgrindingstep(protocolstep2).Thefoursoilswereextractable andgavevariousDNAandRNArecoveryyieldssufficientlyconcentratedtobefurtheranalyzedbyany othermoleculartechnique(Fig.8).Asexpected,themeanRNA/DNAratioobservedfortropicalsoils (0.62)wasgreaterthanthatfortheMediterraneansoilsampledduringthewinter-time(0.17). 5.Additionalinformation

VariationsintheDNAandRNAcompositionbringadditionalinformationasDNAislinkedtocell 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 and20

m

gofDNAand2and5

m

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

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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