OneofthemostproblematicstepsinRNAisolationisthefirststep-thoroughlysisofthetissueorcellsampleinadenaturantsolutionthatinhibitsRNAdegradationbyRNase.WhileitispossIBLetoprocessfreshtissuedirectly,itisextremelyimportantthatallcellsaredisruptedimmediatelyuponcontactwiththedenaturant.Thisusuallyrequiresuseofapolytronandeventhensome"difficulttoprocesstissues"(e.g.hardtumors,bacterialcells,plantroots,etc.)arenoteffectivelydisrupted(seethearticle,"CellDisruption:GettingtheRNAOut").Therefore,ifyouarehavingaproblemwithyieldordegradationduringRNAisolation,weusuallyrecommendfreezingthetissuesamplebeforeprocessing.Herewecomparethreemethodsforprocessingfrozentissuesinaside-by-sidetestforquantityofmRNArecovered.

FreezingtheTissue

Samplesshouldbefrozenquicklysothatthewholetissuesamplefreezesatoncethroughout.Thismaymeanmincingthetissueintosmallerfragmentsbeforefreezing.Submergingthesamplesinliquidnitrogenwillfreezethetissuepiecesmostquickly.Alternatively,ametalplateplacedondryicecanserveasafreezingsurface.

Eachofthemethodsbelowdescribesadistinctwayofgeneratingatissue/celllysatefromwhichtopurifyRNAandisassessedforyieldofpoly(A+)RNA,whenusedtoprocess0.1goffrozenmouselivertissue.Whilethethreemethodseachuseaguanidinebuffertoultimatelylysethecells,theydifferinhowthetissueisprocessedpriororduringthatlysisstep.

Method1:ProcessingfrozentissuefragmentsinadounceYield:4.1µgpoly(A+)RNAFrozentissueiscutintosmallpieces(approx.0.5cm²)ondryice,placedinadounce,andprocessedaslysisbufferisadded.BothpestleAandpestleBareusedfortenstrokeseach.

Method2:ProcessingfrozentissuefragmentsthroughasyringeYield:3.2µgpoly(A+)RNAFrozentissueiscutintosmallpieces(approx.0.5cm²)ondryice,addedtolysisbufferandpassedbackandforthtentimesthroughan18gaugesyringeneedle.

Method3:GrindingthetissuetoapowderinliquidN₂Yield:7.1µgpoly(A+)RNAThefrozensampleispowderedbygrindingthefrozentissuefragmentsinaprechilledmortarandoccasionallyaddingliquidN₂intothemortartopreventthawing.Oncethetissueisgroundtoafinepowder,thedenaturingsolutionisaddedtothemortar,andthesemi-frozenmixtureisstirred.Thismixturecanthenbethawedandtransferredtoanappropriatevesselforfurtherprocessing.

NotethatbygrindingthetissuetoapowderinliquidN₂(Method3),cellulardisruptionismuchmorecompleteresultingalmosttwicetheyieldoftheothertwomethods.