Thesemethodsweresuccessfulinourlabusingprostatetissueandforourspecificobjectives.Investigatorsmustbeawarethattheywillneedtotailorthefollowingprotocolfortheirownresearchobjectivesandtissueunderstudy.

Morethan10,000CellsIftheamountofmicrodissectedmaterialissubstantial(>10,000cells)thenanyofthestandardproceduresforisolatingDNAareacceptable.

Lessthan10,000CellsIfthenumberofcellsprocuredisminimal,e.g.,dissectionofpre-malignantlesions,thenasimpleproteinaseKtreatmentpriortoPCRisrecommended.

ProcuredcellsaresUSPendedinbuffercontaining10mMTris-HCl,1mMethylenediaminetetraaceticacid(EDTA),1%Tween20,and0.1mg/mlproteinaseK,pH8.0,andincubatedovernightat37°C.Longerincubationtimes,higherincubationtemperatures,andhigherconcentrationsofproteinaseKhavebeenreportedtoimprovethequalityofDNArecoveredfromfixedtissuesections.Thesampleissubsequentlyboiledfor8minutestoinactivatetheproteinaseKandtheDNAisreadyforPCRanalysis.

TIP:Ifalesionorparticularcellpopulationcontainingverysmallnumbersofcellsisdesired,itisrecommendedthattheidenticallesionfromasmanyconsecutivehistologicrecutsaspossIBLebedissectedtomaximizethetotalnumberofcellsprocured.

TIP:Thereisnooptimalnumberofcellsthatshouldbecollectedfromamicrodissectionsinceresultsvarysignificantlydependingonthetissuesource.Forfrozentissueapproximately20cells/µlofextractionbufferisrecommendedasagoodstartingpoint;however,morecellsperPCRreactionmaybeneededforDNArecoveredfromformalin-fixed,paraffin-embeddedtissue.

Formalin-fixed,Paraffin-embeddedTissuePCRamplificationofDNArecoveredfromstandardformalin-fixed,paraffin-embeddedtissueispossibleandallowsinvestigatorstoperformstudiesonarchivalpatientmaterial.Studiesonprocessedtissuehavethegeneraladvantagesofabundantsamplesforstudy,highqualityofhistologicdetailforstudyingdysplasticandpre-malignantlesions,andfrequentavailABIlityoffollow-upclinicalinformationonpatients.However,thesestudiesareoftentechnicallychallenging.ThetypeoffixativeusedandthefixationtimeimpactheavilyonthequalityoftheDNArecoveredaftermicrodissection,thusPCRamplificationsignalsmaybewidelydisparateamongsamplesinastudy.

TIP:IfasampledoesnotinitiallygiveaPCRproduct,aten-folddilutionoftheboiledsamplemayyieldastrongPCRproductduetodilutionoftissueinhibitorsofDNApolymerase.

TIP:UtilizationofPCRprimersetsthatproduceproductsinthe150-200bprangeisoptimalsincetheDNAisoftencrosslinkedand/orfragmented,andlargerproductsmaynotbereliablyamplified.

OtherFixativesFixativescontainingheavymetalsorlowpHshouldbeavoided.Non-formalin-basedfixativesgenerallyresultinrecoveryofbetterqualityDNAandmaybecomeincreasinglyimportantforroutineprocessingoftissuesamplesinthefutureasthefieldofmolecularpathologyevolves.

TimingRoutinecareintissueprocessingisalsohelpful,includingimmediateprocessingofsamplesaftersurgeryandslicingoftissuesamplesintothinsectionstoallowrapidpenetrationoffixative.Overfixation(>24hours)shouldbeavoided.

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