• Pipet30mlofRNAProtectBacteriaReagent(Qiagen)intoa50mlpolypropyleneconicaltube.
• Pipet15mlcultureintothetube.Miximmediatelybyvortexingfor5second.Incubatefor5minatroomtemperature.
• Centrifugefor10minat5800g(4500rpmforH-6000ArotorinSORVALLRC-3Bcentrifuge)
• Decantthesupernatant,andleavetubesinvertedonapapertowelfor10s.
• FreezethepelletwithEtOH/DyeIcemix.
• Thepelletcanbestoredat-20°Cupto2weeks,or-70°Cforupto4weeks.

• Dilute2ulofProteinaseKinto300ulofTissueandCellLysissolutionforeachsample.
• ResUSPendcellpelletbytheLysissolutionandmixthoroughly.Transfermixto1.5mltube.
• Incubateat65°Cfor45min,andvortexevery15min.
• Placethesampleonicefor5min.
• Add150ulofMPCproteinPrecipitationReagentto300uloflysedsampleandvortexmixfor10sec.
• Spinfor10min4°Catmaxspeedinamicrocentrifuge.Transferthesupernanttoacleantube.
• Add50ulofMPCproteinPrecipitationReagentandrepeatabovestep.
• Add500ulisopropanoltotherecoveredsupernatant,invertthetube30-40times.
• PellettheRNAbycentrifugationat4°Cfor10mininamicrocentrifuge.
• CarefullypourofftheisopropanolwithoutdislodgingtheRNApellet.Removealloftheresidualisopropanolwithapipet.Airdry10-15min.

• Dilute10ulofRNAse-FreeDNAseIupto200ulwith1xDNAseBufferforeachsample.
• Completelyresuspendthenucleicacidpelletin200ulofDNAseIsolution.
• Incubationat37deg;Cfor45min
• Add200ulof2xTandClysissolution,vortexmixfor5seconds
• Add200ulofMPCreagentvortexmix10seconds,placeonice5min.
• Pelletthedebrisbycentrifugationfor10minat4°C,>10,000ginamicrocentrifuge.
• Add50ulofMPCreagentandrepeat5and6(butthistimespin20min).
• Add600ulisopropanoltotherecoveredsupernatant,invertthetube30-40times.
• PellettheRNAbycentrifugationat4°Cfor10mininamicrocentrifuge.
• CarefullypourofftheisopropanolwithoutdislodgingtheRNApellet.
• Rinsewith75%EtOH(DEPCH₂O),beingcarefultonotdislodgethepellet.Centrifuge5minat4°C
• RemovealloftheresidualEtOHwithapipet.Airdry10-15min.
• Resuspendthenucleicacidin52ulRNAse-freewater.

• Electrophoresison1%Agarosegelwith1ulsample.
• Dilute1ulsampleto100ulwithTE(10mMTris.HClpH8,1mMEDTA),andmeasureA260andA280withaspectrophotometer..Alternatively1ulsamplecanbeusedtomeasureA260andA280onaNanodropND-1000Spectrophotometerwithoutdilution.
• ConcentrationofRNAsample=40xA260x100(Dilutionfactor)(ug/ml)
• A260/A280Ratio=A260/A280,rangingfrom1.7to2.1
• Add100ulEtOH(2volume)and5ul3MNaOAC(1/10volume),storeat-20°C

• RNAProtectBacteriaReagentQiagen
• MasterPureRNAIsolationKitEpicentre

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