Youwillneed:

Guanidineisothiocyanate(Sigma)1Msodiumcitrate,pH7(DEPC-treated,autoclaved)Sarcosyl(N-laurylsarcosine,Sigma)b-mercaptoethanol(Sigma)2Msodiumacetate,pH4(DEPC-treated,autoclaved)3Msodiumacetate,100mMmagnesiumacetate,pH5.2AbsoluteethanolPropan-2-ol(isopropanol)70%ethanol(madewithDEPC-treated,autoclavedwater)0.5%SDS(madewithsterile,DEPC-treatedwater)SolutionD-4Mguanidineisothiocyanate,25mMsodiumcitrate,pH7,0.5%sarcosyl,100mMb-mercaptoethanol.

SolutionDcanbemadeandstoredat4°Cwithoutb-mercaptoethanolforseveralmonths.b-mercaptoethanolshouldbeaddedto100mMimmediatelypriortouse.b-mercaptoethanolisusedtopreventthereformationoftheintra-moleculardisulphidebridges,oneofthereasonsfortheextremestABIlityofribonucleases.

1)TissueishomogenizedasrapidlyaspossIBLe,at4°C,insolutionD(500ulper50mgtissue)withanEppendorfpestlehomogenizeruntilasmooth,lysed,homogenoussUSPensionisobtained.

2)Add50ul2Msodiumacetate,pH4.0andmixvigorously.

3)Add500ulphenolandmixvigorously.

4)Add100ulchloroform,mixvigorouslyandincubateonicefor15minutes.

5)Centrifugemixtureat10,000gfor10minutesinamicrofugeat4°C.

6)Removeupper,aqueousphasetoaclean,sterile,DEPC-treatedeppendorftube.Aftercentrifugation,RNAispresentintheaqueousphasewhile,duetoprotonationattheacidicpHused,genomicDNAispartitionedintothephenolphase.

7)Extracttheupperaqueouslayerwithanequalvolumephenol/chloroformandcentrifugeasbefore.Repeattheextractionsuntilnointerfacematerialisseen.

8)Precipitatetheaqueousphasebytheadditionofanequalvolume(500ul)ofpropan-2-ol.Incubateat-20°Cfor20minutes.

9)PelletRNAbycentrifugationatmaximumspeedinamicrofugefor10minutes.

10)WashtheRNAoncein70%ethanolandvacuumdry.

11)Re-dissolvein200ul0.5%SDSat65°C.

12)Extractwithanequalvolume(200ul)ofphenol/chloroformasabove.Repeatuntilnointerfacematerialisvisible.

13)PrecipitatepureRNAbytheadditionof20ul3Msodiumacetate,100mMacetate,pH5.2and500ulabsoluteethanol.Incubateat-20°Cfor20minutes.

14)PelletRNAbycentrifugationatmaximumspeedinamicrofugefor10minutes.

15)WashtheRNAoncein70%ethanolandvacuumdry.

16)DissolveRNAinappropriatebufferi.e.DEPC-treated,sterileTE,pH8or0.5%SDSifnoenzymicmanipulationoftheRNAisneeded.SDSisaninhibitorofribonucleases.

RNAqualitycanbeassessedbyelectrophoresisunderdenaturingconditionsusingagarose/formaldehydegelsandtheMOPSbuffersystem.