UM-TotalRNA-NucleoZOL

MACHEREY-NAGELENISO9001ENISO13485CERTIFIEDMACHEREY-NAGELGmbH&Co.KG·Neumann-Neander-Str.6–8·52355Düren·GermanyFrance:MACHEREY-NAGELEURLTel.:[phone]E-mail:[e-mail]:MACHEREY-NAGELAGTel.:[phone]E-mail:[e-mail]al:Tel.:[phone]E-mail:[e-mail]:MACHEREY-NAGELInc.Tel.:[phone]E-mail:[e-mail]lNucleoZOLFebruary2016/Rev.02Axxxxx/026x3MACHEREY-NAGEL–02/2016,Rev.02NucleoZOLTableofcontents1Components41.1Kitcontents41.2Reagents,consumables,andequipmenttobesuppliedbytheuser41.3RNase-freeworkenvironment51.4Aboutthisusermanual52Productdescription62.1Thebasicprinciple62.2 Product specifications 72.3Handling,preparation,andstorageofstartingmaterials82.4RNAreconstitution83Storageconditionsandpreparationofworkingsolutions94Safetyinstructions105NucleoZOLprotocols125.1IsolationofsmallandlargeRNAintwoseparatefractions125.2IsolationoftotalRNA155.3IsolationoftotalRNAincombinationwithNucleoSpin®RNASetforNucleoZOL186Appendix206.1DigestionofresidualDNAsolution206.2Troubleshooting216.3Orderinginformation226.4Productuserestriction/warranty23MACHEREY-NAGEL–02/2016,Rev.024NucleoZOL1Components1.1KitcontentsNucleoZOLREF740404.200NucleoZOLreagent200mL1.2Reagents,consumables,andequipmenttobesuppliedbytheuserReagents•RNase-freewater•75 % ethanol•70 % isopropanol•100 % isopropanol•4-bromoanisole(optional,seep.16)Consumables•1.5mL,2.0mLor15mLcentrifugetubes(dependingontheamountofsampletobeprocessedperpreparation)•SterileRNase-freetipsEquipment•Manualpipettors•Vortexmixer•Centrifugeformicrocentrifugetubes•Equipmentforsampledisruptionandhomogenization•Personalprotectionequipment(e.g.,labcoat,gloves,goggles)•Wellventilatedworkingenvironment•RNase-freeworkingenvironment5MACHEREY-NAGEL–02/2016,Rev.02NucleoZOL1.3RNase-freeworkenvironmentThereagenthasbeentestedforfunctionality.However,anRNase-freeworkingenvironmentisalsoacriticalfactorforperformingsuccessfulRNAisolationandhandling.Therefore,generalrecommendationstoavoidRNasecontaminationshouldbefollowed:•Maintainaseparatearea,dedicatedpipettors,andmaterialswhenworkingwithRNA.•WeargloveswhenhandlingRNAandreagentstoavoidcontactwithskin,whichisasourceofRNases.Changeglovesfrequently.•UsesterileRNase-freeplastictubes.TubesforlysatepreparationandRNAprecipitationhavetobesuppliedbytheuser.•Keepallkitcomponentssealedwhennotinuseandalltubestightlyclosedwhenpossible.1.4AboutthisusermanualPlease read the detailed protocol if using NucleoZOL for the first time. Experienced usersmayrefertotheshortinstructionmanual.AlltechnicalliteratureisavailableontheInternetat–02/2016,Rev.026NucleoZOL2Productdescription2.1ThebasicprincipleNucleoZOLisdesignedfortheisolationoftotalRNA(smallandlargeRNA)inasinglefractionorinseparatefractionsfromavarietyofsamplematerials,suchascells,tissue,andliquidsfromhumanoranimalorigin,plants,yeast,bacteria,viralmaterials,andothersources.OneofthemostimportantfactorsduringtheisolationofRNAistopreventdegradation.First,cellsandtissuesarelysedandhomogenizedinNucleoZOLreagentbasedonguanidiniumthiocyanateandphenol.ContaminatingmoleculessuchasDNA,polysaccharides,andproteinsareprecipitatedbytheadditionofwaterandremovedbycentrifugation.TheNucleoZOLprocedureallowstheseparateisolationofsmallandlargeRNAbyaddingethanolandisopropanol,respectively.RNAcanbereconstitutedbyRNase-freewater.Achloroform-inducedphaseseparationisnotnecessaryforhigh-qualityRNAisolation.TheRNAisreadyforuseinqRT-PCR,microarrays,RNaseprotectionassays,polyA+isolation,blotting,andotherapplications.7MACHEREY-NAGEL–02/2016,Rev.02NucleoZOL2.2ProductspecificationsTable1:ProductspecificationsataglanceTechnologyOne-phaseextractionSamplematerial(permLNucleoZOL)1x107culturedcells,bacteria,andyeast,100mghuman/animal/planttissue, 0.4 mL (viral) fluidsFragmentsizeSmallRNA(10–200nt),largeRNA(200nt)Typicalyield(totalRNA)Liver: 6–8 μg/mg tissueKidney, spleen: 3–4 μg/mg tissueMuscle, brain, lung: 0.5–1.5 μg/mgCultured cells: 4–10 μg/106cellsTypicalyield(largeRNA)Liver: 5–7 μg/mg tissueKidney, spleen: 3–4 μg/mg tissueMuscle, brain, lung: 0.5–1.5 μg/mgCultured cells: 3–8 μg/106cellsA260/280(totalRNA)1.8–2.1TypicalRIN(RNAintegritynumber)9ElutionvolumeflexibleMACHEREY-NAGEL–02/2016,Rev.028NucleoZOL2.3Handling,preparation,andstorageofstartingmaterialsSampleharvestandRNaseinhibitionRNA is not protected against digestion until the sample material is flash frozen or disruptedinthepresenceofRNaseinhibitingordenaturingagents.Sampleharvestmethods:•Use freshly harvested sample for immediate lysis and RNA purification.•Samples can be stored in NucleoZOL after disruption at -20 °C to -70 °C for uptooneyear,at4°Cforupto24hours,oruptoseveralhoursatroomtemperature.FrozensamplesinNucleoZOLshouldbethawedslowlybeforestartingwiththeisolationofRNA.•FlashfreezesampleinliquidN2 immediately upon harvest and store at -70 °C. Fr