RNAbased Studies Processing of Microdissected Tissue for Molecular Analysis

Thismethodwassuccessfulinourlabusingprostatetissueandforourspecificobjectives.Investigatorsmustbeawarethattheywillneedtotailorthefollowingprotocolfortheirownresearchobjectivesandtissueunderstudy.

TotalRNAIsolation
RecoveryandanalysisofRNAfromfrozentissuesectionscanbeachievedusingslightmodificationsofstandardmethods.WeobtaintotalRNAfrommicrodissectedsamplesusingtheMicroRNAisolationkit(Stratagene,LaJolla,CA).

Materials:

1. StratageneMicroisolationKit.
2. Beforebeginning,cleanallpipettorswithRNaseawayorasimilarproduct.

Method:

1. PlacetheLCMcaponanEppendorftubecontaining200µlRNAdenaturingbuffer(GITC)and1.6µlb-mercaptoethanol.Invertseveraltimesoverthecourseof2minutestodigestthetissueoffthecap.
2. RemovethesolutionfromthereagenttubeandreplaceitinasturdyRNasefree1.5mltube.
3. Add20µl(0.1Xvolume)2Msodiumacetate,pH4.0.
4. Add220µl(1Xvolume)watersaturatedphenol(bottomlayer;1:1).
5. Add60µl(0.3Xvolume)chloroform-isoamylalcohol.
6. Shakethetubevigorouslyfor15seconds.
7. Placeonweticefor15minutes.
8. Centrifugefor30minutesat4°Ctoseparatetheaqueousandorganicphases.
9. Transferupperaqueouslayertoanewtube.

   TIP:Ifanyofthelowerorganicphasewasaccidentallytransferredandmaybecontaminatingtheaqueousphase,thiswillinterferewiththesubsequentisopropanolprecipitation.Toremoveanyresidualorganicsfromtheaqueouslayer,addonevolumeof100%chloroform,mixwell,andcentrifugefor10minutesat4°Ctoseparatetheaqueousandorganicphases.Transfertheupperlayertoanewtube.

    

10. Addtoaqueouslayer,1-2µlglycogen(10mg/ml)and200-300µlcoldisopropanol(i.e.,equalvolume).

    TIP:Glycogenfacilitatesvisualizationofthepellet,whichcanbeproblematicwhenusingsmallamountsofRNA.

     

11. Placesamplesat-80°Cforatleast30minutes.Itmaybeleftovernight.

    TIP:Beforecentrifuging,thetubesmayneedtobethawedslightlyiftheyhavesolidifiedduringtheisopropanolprecipitation.

     

12. Centrifugefor30minutesat4°Cwithcaphingespointingoutwardsothatthelocationofthepelletcanbebetterpredicted.
13. Removethesupernatantandwashwith300µlcold70%ethanol.Addthealcoholandspinfor5minutesat4°C.
14. Removethesupernatant.
15. Letthepelletairdryonicetoremoveanyresidualethanol.Overdryingpreventsthepelletfromresuspendingeasily.
16. Thepelletmaybestoredat-80°CuntiluseorproceedtoDNasetreatment(below).

OptionalDNaseTreatment
DNAsetreatmentishighlyrecommendedformicrodissectedcells.GenomicDNAcontaminationisoftenproblematicwiththesesamples,possiblyduetothesmallDNAfragmentsthatarecreatedduringtissueprocessingandaredifficulttopurifyfromRNA.

1. ToRNApellet,add15µlDEPCwaterand1µl20U/µlRNaseinhibitor(PerkinElmer).
2. Gentlymixbyflickinguntilthepelletisdissolved.
3. Quickspin.
4. Add2µl10XDNasebuffer(GenHunter)and2µl10U/µlDNaseI(GenHunter;20unitstotal)
5. Incubateat37°Cfor2hours.
6. Re-extractRNAbyadding:

   2µl2Msodiumacetate,pH4.0
   22µlwatersaturatedphenol
   6µlChloroform-isoamylalcohol

    

7. Vortexvigorouslyfor15seconds.
8. Placeonweticefor15minutes.
9. Centrifuge10minutesat4°C.
10. Transferupperlayertoanewtube.

    TIP:Ifanyofthelowerorganicphasewasaccidentallytransferredandmaybecontaminatingtheaqueousphase,thiswillinterferewiththesubsequentisopropanolprecipitation.Toremoveanyresidualorganicsfromtheaqueouslayer,addonevolumeof100%chloroform,mixwell,andcentrifugefor10minutesat4°Ctoseparatetheaqueousandorganicphases.Transfertheupperlayertoanewtube.

     

11. ContinuewithRNAextractionfromStep10inTotalRNAIsolation,adjustingthevolumeofisopropanolaccordingly.