Carl Zeiss MicroImaging PALM Protocols – RNA handling · PALM Protocols - RNA Handling PALM...

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PALM Protocols - RNA Handling

Carl Zeiss MicroImaging

PALM Protocols – RNA handling

PALM Protocols

RNA handlingNon contact Laser Capture Microdissection

Carl Zeiss MicroImaging – Location Munich – Germany

5 Introduction - Some remarks on RNA 6 The DOs and DON’Ts on handling RNA 7 Preparation of slides 7 Samples on MembraneSlide 9 Samples on glass slides 9 Archived samples: removing the coverslip 9 Treatment to remove RNases 9 UV treatment 9 Poly-L-Lysine treatment 10 Mounting samples onto slides 10 Frozen sections 10 Paraffinembedded(FFPE)sections 11 Cytospins 11 Blood and tissue smear 12 Staining procedures 12 Paraffinembedded(FFPE)sections 12 Frozen sections 12 Cresyl Violet 13 Hematoxylin/Eosin(HE) 13 Storage 14 Non-contactLaserCaptureMicrodissection(LCM)Procedures 14 Tips to improve morphological information 14 Diffusor CM 15 AdhesiveCap opaque 15 LiquidCover Glass 16 Collection devices 16 AdhesiveCap 16 Other microfuge tubes 17 Collection procedures 17 “Dry”collection(AdhesiveCap) 17 “Wet”collection(othermicrofugetubes) 17 Capture check - looking into the cap to see the lifted samples 18 Downstream Applications 18 RNA from frozen sections 20 RNAfromFFPEsections 21 UsingcomponentsoftheQIAGENRNeasy®FFPEKit 22 Using other extraction methods 22 Quality control of RNA 23 GeneralremarksonRNA(distribution,content,RNaseactivity) 24 LabTips for working with RNA 25 Otherprotocols(DNA,Chromosomes,LiveCells,...)

Content

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Introduction

Some remarks on RNA

RNAisabiologicalmacromoleculewithmanydifferentfunctions.MessengerRNA(mRNA),transcribedfromDNA,servesastemplateforsynthesis ofproteins.Thisproteinsynthesisiscarriedoutbyribosomes,whichconsistof ribosomalRNA(rRNA)andproteins.

AminoacidsforproteinsynthesisaredeliveredtotheribosomeontransferRNA(tRNA)molecules.RNAsarealsopartofriboproteinsandribozymes.

AnalysisofRNAcanprovideagoodreflectionofanorganism’sgeneexpression profile.Geneexpressionprofilingofmaterialisolatedbymicrodissectionhas become a very important method for analyzing cellular behavior in a micro scale andisusedinresearchandclinicalapplications.

Therefore the isolation of high quality RNA is crucial for all subsequent steps and thesuccessoftheoverallexperiment.

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PALM Protocols - RNA Handling PALM Protocols - RNA Handling

The DOs and DON’Ts of handling RNA

RNAdegradationisacommonreasonforfailingexperiments.RNAisproneto digestionbyawidevarietyofendogenousandexogenousRNases.

These RNases are present on almost any object that comes into contact with human skinandaredifficulttoinactivate.EvenminuteamountsaresufficienttodestroyRNA. Some precautions can make the difference between an intact and degraded RNA prep (seealso:www.ambion.com)andthereforebetweensuccessfulandunsuccessfulexperiments.

DOs

• designateaspecialareaforworkingwithRNA• cleanbencheswithspecialcleaningsolutionse.g.RNaseZap(AMBION,#9780)• wearglovesandchangethemfrequently• usesterile,disposableplasticware• glasswareshouldbetreatedwith0.1%DiEthylPyroCarbonate(DEPC) or oven baked at 180°C for at least 4 hours before use• usepipettetipswithfilters• aqueoussolutionsshouldbetreatedwith0.1%DEPC• useonlyRNase-freereagents,tubesandtips• forbestresultsusesamplesthathavebeensnapfrozenondryiceorinliquidnitrogen; all required reagents should be kept on ice• storepreparedRNA,aliquotedinethanolorRNAelutionbufferat-80°C• toavoidcondensationofmoistureduringthawing,theslidesshouldbefrozen at-80°Candrethawedinatightlysealedcontainer(e.g.50mlFalcontube)• ingeneraluseprotocols(e.g.staining)withshortincubationtimesonice

DON’Ts

• don’tbreathonsamples;someresearcherswearmasks• don’ttouchanythingwithbarehands• don’tautoclavepipettetips,aswatervapormaycontainRNases• don’tallowfrozentissuetothaw• don’tresuspendRNAinDEPCwater;residualDEPCcaninhibitdownstreamreactions


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Preparation of slides – Samples on MembraneSlide

MembraneSlides are special slides covered withamembraneononeside. This membrane is easily cut together with the sample and acts as a stabilizing backbone duringlifting.Thereforeevenlargeareas are lifted by a single laser impulse without affectingthemorphologicalintegrity. Use of MembraneSlide is especially important forisolatingsinglecells,chromosomesaswellaslivecellsorsmallorganisms.

CarlZeissMicroImaging(CZMI)offersslides(1mm,0.17mm)coveredwithpolyethylenenaphthalate(PEN)-membrane.ThisPEN- membrane is highly absorptive in the UV-A range,whichfacilitateslasercutting.The membrane can be used for all kind of applications.When working with low magnifying objec-tives like 5x or 10x both regular 1 mm thick glassslidesand0.17mmglassslidescanbeused.Tokeepthisflexibilityforhighermagni-fications(20x,40xor63x)CZMIrecommendsusinglongdistanceobjectives.

With those objectives you have the possibility to adapt the working distance to the different glass slides by moving the correction collar on theobjective.

Due to the short working distance of the 100xmagnifyingobjectivesonly0.17mm thincoverglassslidescanbeused.

MembraneSlideNF(nucleasefree)iscertifiedtobefreeofDNase,RNaseandhumanDNA.InadditiontoPEN-MembraneSlide,CZMI alsoofferspolyethyleneteraphthalate(PET)-membranecoveredslides.Theseslidesarehelpfulforspecialprocesses,i.e.forisolation ofchromosomesandsomefluorescenceapplications. AlternativelythePET-membraneisavailable attachedtoametalframe(FrameSlidePET). Influorescenceapplications(FISH)evenweaksignals can be detected due to low signal to noiseratio.TheframestructureofFrameSlidePETisresistanttomicrowavetreatment.Thespecial bonding is inert and adapted to heat treatment(upto95°C)sothatthemembranedoesnotruffleduringtheheatingprocess.Ifyouneedinformationabouttheseslides,please contact:

E-Mail: [email protected]

Regular glass slide (1 mm thick) => 1, thin slide (0.17 mm thick) => dot,

DuplexDish and FrameSlide => between dot and 0.


MembraneSlide 1.0 PEN - Order No. 415190-9041-000 (white)MembraneSlide 1.0 PEN NF - Order No. 415190-9081-000 (white)MembraneSlide 0.17 PEN - Order No. 415190-9061-000 (uncolored)MembraneSlide 50x1.0 PEN - Order No. 415190-9091-000 (doublewidth)MembraneSlide 1.0 PET - Order No. 415190-9051-000 (blue) FrameSlide PET - Order No. 415190-9101-000 (metal)

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Preparation of slides – Samples on glass slides

With PALM MicroBeam almost every kind of biological material can be microdissected andlifteddirectlyfromglassslides. Evenarchivalpathologicalsectionscanbeused after removing the cover slip and the mountingmedium.

To facilitate easy lifting additional adhesive substances or “Superfrost + charged slides” should only be applied when absolutely necessary for the attachment of poorly adheringmaterial(e.g.somebrainsectionsorbloodvesselrings).In those cases higher laser energy is needed forlifting.

From the dry glass slide sample material can be lifted directly by “AutoLPC” function ofPALMRoboSoftware.

Treatment to remove RNases

Treatment of MembraneSlides and glass slidestoremoveRNasesareidentical.Slides are shipped without any pretreat-ment.• ToensureRNase-freeMembraneSlides orglassslides,heatslidesat180°C for4hourstocompletelyinactivateRNases.•MembraneSlideNF(nucleasefree)is certifiedtobefreeofDNase,RNaseand humanDNA. Treatments to remove nucleases are there- forenotnecessaryusingtheseslides.

UV treatment

To overcome the hydrophobic nature of the membrane it is advisable to irradiate with UV light at 254 nm for 30 minutes (e.g.inacellculturehood). Themembranegetsmorehydrophilic, thereforethesections(paraffin-andcryosections)adherebetter.Positivesideeffects are sterilization and destruction ofpotentiallycontaminatingnucleicacids.

Poly-L-Lysine treatment

Additional coating of the slide with Poly-L-Lysine(0.1%w/v,e.g.SIGMA,#P8920)only will be necessary for poorly adhering materials(e.g.brainsections)andshouldbeperformedafterUVtreatment. Distribute a drop of the solution on top of theslide. Let air-dry at room temperature for 2-3 minutes.Avoidanyleakageofthemem-brane,asthismightresultinimpairment ofLaserCaptureMicrodissection.

Archived samples: removing the coverslip

Depending on the applied mounting medium(whetheritissolubleinxyleneorwater)thewholeslideshouldbecompletely submerged in the respective solvent.

1.standingupinaglassjarfilledwith either pure xylene or warm water (30-50°C)

2. timeneededforthecoversliptoswim off may range from hours to days

3.gentlemovementofthejarmayspeed up the process

4.air-drytheslideafterremoval

Note: It is very important NOT to use any force to push off the coverslip because this might damage the section! Wait till it falls off by itself! The necessary time depends on the age of the sample and the dryness ofthemountingmedium.Freshslides(onlydaysold)canbedecover-slippedmuchfaster.

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Mounting samples onto slides

Frozen sections

SectioningSections are mounted onto Membrane- Slides the same way as routinely done usingglassslides.Toallowsubsequent cutting and lifting a coverslip and standard mountingmediummustnotbeapplied.Freezing media like OCT or similar may be used but should be kept to a minimum and havetoberemovedbeforelasercutting.

For optimal RNA protection take a pre-cooled slide and touch the backside of the slidewithyourfinger(gloves!)towarmonlytheregionforplacingthesection. Now transfer section from the knife by touching with the warmed area and dry at-20°Cinthecryostatfor2-3minutes.

FixationCZMIrecommendsthedehydrationin ice-cold70%ethanolfor2-3minutes.

Removing the tissue freezing mediumIf OCT or another tissue freezing medium isused,itisimportanttoremoveitbefore LaserMicrodissection,becausethesemedia willinterferewithlaserefficiency. Removing the medium is easily done by dipping the slide 5-6 times in ice-cold RNase-freewater.Ifthesectionsarestai-nedinaqueoussolutions,thesupportingsubstance is normally removed “automa-tically”bythewatercontainingsteps.

Formalin Fixed Paraffin Embedded (FFPE) sections

SectioningSections are mounted onto MembraneSlides the same way as routinely done using glass slides.Floatingthesectiononwarmwater aswellashotplatetechniquescanbeapplied.Aftermounting,letdrytheslides overnight inadryingovenat56°C.Toallowlasercutting and lifting a coverslip and standard mountingmediummustnotbeapplied.

DeparaffinationParaffinwillreducetheefficiencyofthe laser,sometimescompletelyinhibiting cuttingandlifting.Ifyouareworkingwith unstained sections it is therefore very importanttoremovetheparaffinbefore lasercuttingandlifting. If applying standard staining procedures deparaffinationisroutinelyincludedinany protocol.1mmMembraneSlidescanbe usedlikenormalglassslides.

Minimal procedure:

1.Xylene2minutes,2times

2.Ethanol100%1minute

3.Ethanol96%1minute

4.Ethanol70%1minute


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Blood and tissue smear

Distributeadropof(peripheral)blood ormaterialofasmearovertheslide. Be careful to avoid injuries in the mem-brane,whichwouldleadtoleakage duringfixationorwashingstepsand therefore would impair the laser capture microdissectionprocess.Letsmearsair-dryshortlyandfixthem for2upto5minutesin70%ethanol.

Cytospins

Cytospins can be prepared on glass slides oronMembraneSlides.Aftercentrifugationwithacytocentrifugeletthecellsair-dry.Thenfixfor5minutesin100%methanol.Allow the cytospins to dry at room tempe-raturebeforestaining.

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Staining procedures

For isolation of high quality RNA use only freshly prepared and precooled staining solutionsandtakenoticeofourtipsonhandlingRNA(pleaseseepage24).

Formalin Fixed Paraffin Embedded (FFPE) sections Cresyl Violet

Frozen sections

Afterdeparaffinationcontinuewiththe stainingprocedureofyourchoice. Most standard staining procedures can beusedforFFPEsections(forrecommen-dationsseeFrozensections).

Moststandardhistologicalstainings(e.g.,HE,MethylGreen,CresylViolet,NuclearFastRed)arecompatiblewithsubsequentRNAisolation.

Note: Using frozen sections endogenous RNases may still be active after the short fixationstep.Thereforeitisrecommededto keepallincubationstepsasshortaspossible. Please use RNase-free water and solutions forallsteps.Allrequiredreagentsshouldbekeptonice.

AtZEISSLabsweusuallyperform theCresylVioletorHematoxylin/Eosin(HE)staining.

This short staining procedure colors the nucleivioletandthecytoplasmweakviolet.It is recommended for RNase-rich tissues since all solutions contain high ethanol concentrations.

Procedure

1.afterfixation(2min,70%Ethanol) dipslidefor30secinto1%cresylviolet acetatesolution(*)

2.removeexcessstainonabsorbent surface

3.dipinto70%Ethanol

4.dipinto100%Ethanol

5.air-dryshortly(1-2min)

(*) Dissolvesolidcresylvioletacetate (e.g.ALDRICH#86,098-0)atacon- centrationof1%(w/v)in50%EtOH at room temperature with agitation/ stirringforseveralhourstoovernight. Filter the staining solution before use toremoveunsolubilizedpowder. (SometimesLottoLotvariationsinthe purchased cresyl violet powder can lead to weaker staining results if the dye contentisbelow75%).


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Hematoxylin/Eosin (HE)

HE-stainingisusedroutinelyinmosthisto- logical laboratories and does not interfere with good RNA preparation if intrinsic RNaseactivityislow.Thenucleiarestained blue,thecytoplasmpink/red.

Procedure

1. afterfixationquicklydipslide5-6times in RNase-free distilled water

2. stain 1-2 minutes in Mayer’s Hema- toxylinsolution(e.g.SIGMA,#MHS-32)

3. rinse1minuteinDEPC-treatedtap waterorblueingsolution(BBC,#3900)

4. stain10secondsinEosinY (e.g.SIGMA,#HT110-2-32)

5. perform a quick increasing ethanol series(70%,96%,100%)

6. air-dry shortly

Storage

Stained slides can be used immediately or storedat-80°CbeforeLCM.To avoid excess condensation of moisture duringthawing,theslidesshouldbefrozeninatightlysealedcontainer(e.g.twoslidesbacktobackina50mlFalcon-tube). For rethawing the container should not be opened before it is completely warmed up againtoambienttemperature.

Note: In most cases this cresyl violet stainingprocedurewillbesufficientfor cellidentification.Ifanenhancementof thestainingisdesired,areinforcement bytwoadditionalstepsin50%ethanol(firstbeforethestainingincresylviolet;secondafterthestainingincresylviolet) ispossible.Additionalintensificationcan be obtained by increasing the working temperature of all solutions to room temperature.The endogenous RNase acitivity varies betweendifferenttissues(pleasesee page23).Therefore,whentheshortstainingprotocolismodifiedbyadditionalsteps(50%ethanol)orbyincreasing theworkingtemperatureZEISSLabsstron-gly recommend a quality control of the RNA(pleaseseepage22).

AmbionofferstheLCMStainingKit(#1935)whichalsocontainsacresylvioletdye.

When using this kit we recommend to omitthefinalxylene-stepoftheAmbion instruction manual because xylene makes the tissue very brittle and reduces the ad-hesionofthesectiontothePEN-membrane.

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Non-contact Laser Capture Microdissection (LCM) ProceduresPlease,additionallyhavealookintothePALMMicroBeamusermanual.

Tips to improve morphological information

EmbeddingandglasscoveringofthespecimenisinapplicableforLCM. Thus,theroughopensurfaceofthesection/materialoftenresultsin impairedviewofmorphology.

Diffusor CM

Diffusor CM can be inserted into PALM Cap- Mover like any holder and is moved over thesample.Theopaqueglassdiffusestheincidentmicroscopelight,whichsmoothens theharshnessofcontrastand,dependingonmaterialandstaining,evenminute details as nuclei and cell boundaries show up.Evenslightdifferencesincolorbecomevisible.Formoredetailsandhandling,please seeDiffusorCMproductinformation.Holders for PALM RoboMover and PALM CapMoverIIareequippedwithdiffusors.

Diffusor CM - Order No. 415101-2100-320PALM CombiSystem


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AdhesiveCap opaque - Order No. 415190-9201-000 (500 µl) AdhesiveCap opaque - Order No. 415190-9181-000 (200 µl)

Liquid Cover Glass

The polymeric and low viscose Liquid Cover Glass completely embeds the tissue and smoothenstheroughtissuesurface,resulting inenhancedmorphology.

Formoredetailsandhandling,pleaseseeLiquidCoverGlassproductinformation.

AdhesiveCap opaque

Thewhite/opaquefillingofAdhesiveCapclearly improves visualization of morpho-logical information of the samples at the object plane due to enhanced color ba-lanceandcontrast,whichmakestheviewcomparable to those of coverslipped tissue sections.Two different microfuge tube sizes with thesefilledcapsareavailablefromCZMI.

Formoredetailsandhandling,pleaseseeAdhesiveCapproductinformation.

Liquid Cover Glass - Order No. 415190-9020-000

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Collection devices

AdhesiveCap

The intention of AdhesiveCap is to allow LCM(LaserCaptureMicrodissection)with-out applying any capturing liquid into the capspriortoLCM.ThisminimizesRNaseactivity.Beside the quick relocation of the lifted samples in the cap due to instant immo-bilization there is no danger of evaporation and crystal formation during extended specimenharvesting.Formoredetailsandhandling,pleaseseealsoAdhesiveCapproductinformation.

Note:CZMIrecommendsAdhesiveCapasacollectiondeviceforallRNAexperiments.

Other microfuge tubes

Other commercially available RNase-free plasticwarecanbeused,too. (e.g.ABgene#AB-0350;0.5mltubes)

If there are no RNase-free tubes available use the following procedure to remove RNasesfromregulartubes.

Treatment of microfuge tubes to remove RNases

add0.1mlDEPCto100mlofdouble distilledwatertogeta0.1%DEPC solution(DEPC:e.g.ROTH#K028.1)

1.stirfor5-6hatroomtemperatureto dissolvetheDEPC

2.soakthereactiontubesintotheDEPC solution,takecarethatthetubesare completely covered with liquid (notblistered!)andincubateovernight at room temperature

3.autoclavethetubestogetherwith the solution for 20 minutes at 121°C toinactivatetheDEPC

4.discardtheliquidcarefullyand thoroughly.Drythetubesat50°C-80°C

5.usethetubesasusual

Note:DEPCistoxicandshouldbeused under a hood!

AdhesiveCap opaque - Order No. 415190-9201-000 (500 µl) AdhesiveCap opaque - Order No. 415190-9181-000 (200 µl)

AdhesiveCap clear - Order No. 415190-9211-000 (500 µl) AdhesiveCap clear - Order No. 415190-9191-000 (200 µl)


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Collection procedures

PleasehavealookintothePALMMicroBeamusermanual.

„Dry“ collection (AdhesiveCap)

Note:CZMIrecommendsAdhesiveCapas collectiondeviceforallRNAexperiments. Capturing without liquid minimizes RNase activity.

After LCM add a lysis buffer of your own choice(e.g.QIAGEN:350µlRLTbuffer) and incubate „upside down“ for 30 minutes.

Subsequently centrifuge the lysate and then apply the routine RNA extraction procedure.

“Wet” collection (other microfuge tubes)

Pipette20µllysisbufferintothecap. The lifted cells or cell areas will stick onto the wet inner surface of the cap and will not fall down after the lifting procedure.Be aware that aqueous solutions will dryoutafterawhile.When using glass mounted samples it may be advisory to put more liquid (upto40µl)intothecap.

Capture check – looking into the cap to see the lifted samples

Tocontroltheefficiencyofliftingitis possible to have a look into the collection device(e.g.microfugecap)withthe5x,10x,20x,40xand63xobjectives. By using the software function “go to check- point” the slide is moved out of the light path and the cap can be lowered further towardstheobjectivesforlookinginside.

„Dry collection“ procedure

Note: Please do not use any water bath fortheupsidedownincubation.

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Downstream Applications

RNA from frozen sections

For RNA extraction a procedure of choice canbeused.To capture microdissected samples from frozensectionsZEISSLabsrecommendAdhesiveCap.

TheRNeasy®MicroKit(QIAGEN,#74004)combinedwithAdhesiveCap(500µl)inourhands results in very good yield and quality ofRNA. (Forrecommendedmodificationstothe originalQIAGENprotocolpleaseseepage19)

ThefinalRNAsolution(12µl)maybestored at -20°C or used directly for reverse trans-cription.QualitycontrolbydirectanalysisliketheAgilentBioanalyzer(RNA6000PicoLabChip®Kit)islimitedtoconcentrationsabove50pg/µlandmayonlybepossiblewithlargemicrodissectedsamples(some2mm2 of collected areas from tissue sections of5-10µmthickness).

Wenormallyuse5to10µlofthefinalRNAsolutionastemplateinaRT-reactionof20µl (e.g.,TranscriptorFirstStrandcDNASynthesis Kit,ROCHE,#04379012001).


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Applying the components of the QIAGEN RNeasy® Micro Kit:

1. Add350µlBufferRLTcontainingß-MercaptoethanoltothetubewiththeLCM elementsintheAdhesiveCap,thenvortexandincubateinan“upsidedown” positionfor30min. ThoroughlysisisessentialforgoodRNAyield.

Note: ß-Mercaptoethanol(ß-ME)mustbeaddedtoBufferRLTbeforeuse. Add10µlß-MEper1mlBufferRLT.Dispenseinafumehoodandwear appropriateprotectiveclothing.BufferRLTisstableatroomtemperature for1monthafteradditionofß-ME.

2. Spindownthelysateinamicrocentrifugefor5minutes. (13400rcf;e.g.Eppendorf5415D:12000rpm)

Note: Samples can now be stored for later use at -80°C or extracted immediatelyfollowingtheoriginalprotocoloftheQIAGENRNeasy® MicroKit(Handbook04/2003).

3. TocontinuewiththeisolationtransferthelysatetoaRNase-free1.5ml microcentrifugetube.

4. Nowswitchtostep5oftheQIAGENprotocol“TotalRNAIsolationfrom MicrodissectedCryosections”(RNeasy®MicroHandbook04/2003,pp20).

5. “Add1volume(350µl)of70%ethanoltothehomogenizedlysate,andmix wellbypipetting.Donotcentrifuge.Continueimmediatelywithstep6.”

Note: Allfurthersteps(6-14)oftheQIAGENprotocolremainunchanged andshouldbeperformedstepbystepaslistedthere. PleaseconsideralsothecommentsandtipsoftheQIAGENRNeasy® manual,especiallythesection:“Thingstodobeforestarting”

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RNA from FFPE sections

ForcollectingmicrodissectedsamplesZEISSLabsrecommendAdhesiveCap.

ZEISSLabsprefertheQIAGENRNeasy®FFPEKit(#74404)withsomespecificmodifica-tions.

This procedure is very effective and allows ahighfinalconcentrationofRNAduetoasmallelutionvolume.GenomicDNAcon-tamination is minimized by a special DNA removalcolumn(gDNAEliminatorspincolumn).Since normally only stained tissue sections areusedformicrodissectionthedeparaffi-nation and staining is done according to standardproceduresforslides(pleaseseepages10,12and13).Furthermore,theincubationwithProteinaseK inPALMprotocolsisprolongedsignificantly comparedtotheQIAGENRNeasy®FFPEprotocol,becauseallourtestswithlasermicrodissected material from various tissues showed higher RNA yields applying longer digestiontimes.

Note:ForformalinfixedsamplesaPro-teinaseKdigestionstepisessential. ThetimenecessaryforoptimalProteinaseK digestion depends on many factors like tissuetype,fixationprocedureorelementsizeofliftedmaterial.Anovernightdigestion (12-18hours)isagoodstartingpointforoptimization but shorter digestion times maybetestedaswell.Toourexperienceatleast 3 hours digestion should be applied withanyextractionprocedureandmaterial.The RNA solution may be stored at -20°C oruseddirectlyforreversetranscription.Quality control by direct analyses like the Agilent Bioanalyzer is very limited and only possible with large microdissected samples (some4mm2fromtissuesectionsof5-10µmthickness).

Wenormallyuse5to10µlofthefinal RNAsolutioninaRT-reactionof20µl (e.g.,TranscriptorFirstStrandcDNA SynthesisKit,ROCHE,#04379012001)usingrandom-oligomers(insteadofoligoT)asprimersforthecDNAsynthesis.

Note:Theuseofrandomorgene-specificprimersisimportant.Reversetranscription offormalinfixedRNAwithstandardoligoT-primersisinefficientandstrongly3-prime biased due to the numerous strand breaks andmodificationsinflictedbytheformalinfixationandparaffinembeddingprocedure.


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Using components of the QIAGEN RNeasy® FFPE Kit:

1. Add150µlBufferPKDand10µlofProteinaseKtothetube,containingthe LCMelementsintheAdhesiveCap,andvortexinan“upsidedown”position.

2. Useanincubatortodigestthesamplesinan“upsidedown”positionat55°C overnight(orforatleast3hours),thenvortexandheatat80°Cfor15minin aheatingblock.

3. Add320µlofBufferRBCtoadjustbindingconditions.

4. MixthelysatethoroughlyandtransferittoagDNAEliminatorspincolumn placedina2mlcollectiontube.Centrifugefor30secat≥8000xg(e.g. Eppendorf5415D:≥10000rpm).Discardthecolumnandsavetheflow-through.

5. Add720µlof100%ethanoltotheflow-throughandmixwellbypipetting. Donotcentrifuge.Proceedimmediatelytothenextstep.

6. Transfer700µlofthesampletoaRNeasyMinElutespincolumnplacedina 2mlcollectiontube. Closethelidgentlyandcentrifugefor15secat≥8000xg(≥10000rpm). Discardtheflow-through.Reusethecollectiontubeinstep7.

7. Repeatstep6untiltheentiresamplehaspassedthroughtheRNeasyMinElute spincolumn.Reusethecollectiontubeinstep8.

8. Add500µlBufferRPEtotheRNeasyMinElutespincolumn.Closethelidgently andcentrifugefor15secat≥8000xg(≥10000rpm)towashthespincolumn membrane.Discardtheflow-through.Reusethecollectiontubeinstep9.

Note: BufferRPEissuppliedasaconcentrate.Ensurethatethanolisadded toBufferRPEbeforeuse.

9. Add500µlBufferRPEtotheRNeasyMinElutespincolumn.Closethelidgently andcentrifugefor15secat≥8000xg(≥10000rpm)towashthespincolumn. After centrifugation carefully remove the spin column from the collection tube so thatthecolumndoesnotcontacttheflow-through.

10. PlacetheRNeasyMinElutespincolumninanew2mlcollectiontube,anddiscard theoldcollectiontubewiththeflow-through.Openthelidofthespincolumn andcentrifugeatfullspeedfor5min. Discardthecollectiontubewiththeflow-through.Itisimportanttodrythespin columnmembrane,sinceresidualethanolmayinterferewithdownstreamreactions.

11. PlacetheRNeasyMinElutespincolumninanew1.5mlcollectiontube. Add14-30µlRNase-freewaterdirectlytothespincolumnmembrane. Closethelidgentlyandcentrifugefor1minatfullspeedtoelutetheRNA. ThedeadvolumeoftheRNeasyMinElutespincolumnis2µl:elutionwith14µl ofRNase-freewaterresultsina12µleluate.

12. TheRNAsolutionmaybestoredat-20°Coruseddirectlyforreversetranscription.

Note: QualitycontrolbydirectanalysisliketheAgilentBioanalyzer(Picochip)is very limited and may only be possible with quite large microdissected samples(oftensome4mm2collectedareafromtissuesectionsof5-10µm thickness).

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Using other extraction methods

ApartfromtheQIAGENKittherearemanyother possibilities and kits to extract RNA fromFFPE-material.Dependingonthe material and the experience of the user even simple procedures like homemade AGTC-methods or Trizol can be quite efficient.IftheoriginalextractionprotocoldoesnotcontainanyProteinaseKdigestion step we recommend to apply a simple procedureaslistedbelow.

Procedure

1.Add20µldigestionbuffercontaining ProteinaseK(150mMNaCl,100mMTris pH7.5,0.5%Igepal,0.5µg/µlProteinaseK) to the tube containing the LCM elements intheAdhesiveCap.

2.Useanincubatortodigestthesamplesin an “upside down” position at 55°C overnight.

3.Spindownthelysateinamicrocentrifuge. (13400rcf;e.g.Eppendorf5415D:12000rpm)

4.InactivateProteinaseKbyheatingto 90°Cfor10minutes.

5.Addtheappropriatelysisbufferandmix byintensevortexing;ifnotproceeding immediately,storethedigestedsamples at-20°Cor-80°C.

6.Continuewithyourpreferredextraction procedure.

Note:ProteinaseKdigestiontimeshouldbeoptimizedforanytissuesample(atleast3hoursarerecommended,butupto18hoursmaybemoreefficient).

Quality control of RNA

The most common method used for assessing the integrity of total RNA is to analyze the RNA sample on an agarosegel.Ingeneral,atleast200ng ofRNAmustbeloadedontothegel.

To analyze RNA samples with concentrations downto50pg/µl,theAgilent2100Bioana-lyzer is an alternative to traditional gel-based analysis and provides information about RNAquality(degradation,purity)andquan-tity(seealso:www.chem.agilent.com).

A prognosis of the expected amount of RNA inatissueisdifficultsincemanyfactors likespecies,cell/tissue-type,fixation, staining,fragmentation,extractionproce-dureandotherswillinfluencetheoutcome.


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General remarks on RNA (distribution, content, RNase activity)

Atypicalmammaliancellcontains10-30pgtotalRNA(mRNA,rRNA,tRNA).Themajorityof RNAmoleculesaretRNAsandrRNAs.mRNArepresentsonly1-5%ofthetotalcellularRNA.

Approximately360000mRNAmoleculesarepresentinasinglecell,correspondingto approximately12000differenttranscriptswithatypicallengthof2kb.SomemRNAs compriseasmuchas3%ofthemRNApoolwhereasothersaccountforlessthen0.01% (QIAGEN,Benchguide).

Also the RNase activities vary dramatically across differenttissues(KrostingJ,LathamG,AMBION,Inc.). A comparison of total RNase activities for 8 different mouse tissues showed that total RNase activity spans a181,000-foldrangefrompancreastobrain,which pointsouttheimportanceofRNasecontrol.

RNA distribution in a typical mammalian cell

Total RNA per cell ~ 10-30 pg 80-85% rRNA(28S,18S,5S) 15-20% tRNAs,snRNAs,lowMWspecies 1-5% mRNAs

Total RNA in nucleus ~14%

DNA : RNA in nucleus ~ 2:1

mRNA molecules 2x105 - 1x106 per cell

Typical mRNA size 1900 nt

RNA content in various cells and tissues

Total RNA mRNA (µg) (µg)

Cell cultures (107 cells) NIH/3T3 120 3 HeLa 150 3 COS7 350 5

Mouse tissue (100 mg) Brain 120 5 Heart 120 6 Intestine 150 2 Kidney 350 9 Liver 400 14 Lung 130 6 Spleen 350 7

Quantitative hierarchy of RNase activity in mouse tissues (AMBION, Inc.)

Mouse tissues Fold increase relative to brain

Pancreas 181,000Spleen 10,600Lung 5,300Liver 64Thymus 16Kidney 8Heart 2Brain 1

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ZEISS Labs: Tips for working with RNA

ForbestRNAqualityweusefrozensectionsonMembraneSlides.Frozensectionsshouldnotbestoredformorethanafewdaysat-80°C.Afterstaininganddryingfreezingshouldbeperformedinanairtightcontainer.AprognosisoftheexpectedamountofRNAisdifficultsincemanyfactorswillinfluencetheout-come(seeabove).Frommouseliverfrozensectionsweusuallyareabletoretrieve5-20pgRNApercell(calculatedfromextractionsof1000cellsandanalysiswithanAgilentBioanalyzer;AgilentApplicationNote5988-ENonourwebsiteoratwww.chem.agilent.com).

Note: Quantitative results from an analysis with the Agilent Bioanalyzer RNA Pico kit are dependentonthesaltcontentofthesample.

Archivaltissuesaremostlyformalin-fixedandparaffin-embedded.RNAextractionfromthesetissues isnotveryeffectivebecauseofthecrosslinkingpropertiesofaldehydes.Othermethodologiesfor preservationofhighmolecularweightRNAinFFPEtissuearedescribedby:

Vinceketal.2005(DiagnMolPathol,14,3:127-133)and Olertetal.2001(PatholResPract,197:823-826)

Formoreinformationseeourwebsite:www.zeiss.de/publications

Summarized recommendations:

•KeepattentiontoDOsandDON´TsonhandlingRNA(page6)

•TakeAdhesiveCapascollectiondeviceforallRNAexperiments(page16)

•Chooseashortstainingprocedurefortissueswithhighcontentof endogenousRNases(e.g.CresylViolet)(page12)

•RNeasy®MicroKit(QIAGEN,#74004)resultsingoodRNAyield (qualityandquantity)fromfrozensectionsinourlab(page18)

•RNeasy®FFPEKit(QIAGEN,#74404)resultsingoodRNAyield (qualityandquantity)fromFFPEtissueinourlab(page20)


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Brochures and protocols

Live cells Chromosomes DNA

FISH Immunofluorescence RNA

Forquestions,commentsorprotocolrequestspleasecontact:

ZEISS Labs

E-Mail: [email protected] Hotline: +49 8990 9000 900

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Aug

ust

2011

PA

LM

Pro

tocols

– R

NA

handling

Carl Zeiss MicroImaging GmbH07740 Jena, Germany

BioSciences I Location MunichPhone : +49 8990 9000 800Telefax: +49 8990 9000 820E-Mail : [email protected]

www.zeiss.de/microdissection

For more scientific details please visit:[email protected]

For scientific questions please contactE-Mail: [email protected]: +49 8990 9000 900www.zeiss.de/labs

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