RNA isolation for Microarray

DescriptionRNAextractionusingTRIREAGENT.ThismethodgivesampleamoutofRNA.

ProcedureItis3daysprocedure.

Day1:

1.Harvestthecellsandcentrifugeat2000rpmfor5-10minutesandpouroffthemedia.2.WashthecellpelletwithPBSandadd10mlTriReagentsolution.3.IncubateatRoomTemperaturefor5minandduringincubationpassthecellsuspensionthroughPipette.4.Collectthesolutionsinfresh50mltubesandincubateatRTfor5min(toensurenucleoproteincomplexdissociation).5.Add1mlof1-Bromo-3-chloropropane(BCP)(forper10mlTriReagent)andshakevigorouslyfor15sec.6.IncubateatRTfor2-15minutes(Average10minutes).7.Centrifugehomogenateat3200rpmfor30minto1hrat4oc.8.Aftercentrifugationtherewillbetwophase:-LowerredPhenolphaseanduppercolorlessRNAaqueousPhase.9.Transferupperaqueousphasetofreshtubebutleavelower1mlofaqueousphase.10.Add5mlofisopropanol(per10mlofTriReagent)11.Shakevigorouslyandleaveat-20oCovernight.

Day2:1.Centrifugeat3,200rpm(12,000g)for30min-1hrat4oC.Takeoffmostofwaterphasebypipette,leaveabout1mlinthetube.2.Add10mlof70%ice-coldethanol(fromthefreezer!)/10mlofTriReagentused.3.IncubatetheRNAat-20oCfor30minutes.4.CentrifugeRNAat3,200rpm(12,000g)for15minat4oC.5.Carefullypourofftheethanol,add1ml70%ethanolintotubesandgentlyflickthetube.6.TrytocollectallRNA,mightaddadditional0.5ml70%ethanoltowashthetube.7.TransferthisRNAintoeppendorftube,resuspendcarefullyandspinitagaininmicrocentrifugeat4oCat11500rpmfor30minutestosedimentRNA.8.Pouroffethanol;tiltthetubedownwardtoallowdrainexcessofethanol.Donotdry!9.UseRNAasefreefilterpaper(soakedinethanolanddriedunderthehood).DonottouchtheRNApellet.10.Add250ulofHPLCgradewater.DissolveRNAandallowittostayatRTfor5min.11.Add25ulof3MSodiumacetatebufferandthen625ulof100%ethanolfromthefreezer.12.Leaveovernightinthefreezerat-20oCtoprecipitate.

Day3:

1.SpinRNAagaininmicrocentrifugeat40C(10min)tosedimentRNA.2.Pouroffethanol;tiltthetubedownwardtoallowdrainexcessofethanol.UseRNAasefreefilterpaper(soakedinethanolanddriedunderthehood).Dryprecipitatefor5min.3.ResuspendtheRNApelletin250ulofHPLCgradewater.Placeonice.4.DonotshakevigorouslyofpipetteRNA.5.DetermineconcentrationofRNAcontent.6.Readabsorptionat260and280inspectrophotometer.7.Quantitativeanalysis:Abs260x400x40=amountofRNAinug/ml

RecipesTriReagent(MRC)1-Bromo-3-chloropropane(BCP)(MRC)3MSodumacetatebuffer(Sigma)isopropanol100%ethanolHPLCgradewater

Supplies

Tips

Submittedby:sbafna