RNA Analysis Notebook .pdf

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1、Microarrays...PurifyRNAwithRNAgents®SystemPurifyprobeplasmidlibrarywithMagneSil®Plasmidor∑Wizard®SV96PlasmidLabelwithImProm-IITMRT∑SystemAmplifyprobeswith∑PCRRemoveRNAMasterMixwithRNaseH∑∑PurifyPurifyzamplimerswithMagneSil®PCRClean-Upor∑Wizard®HybridizeSV96PCRClean-U

2、p∑SeeAnalyzingRNAProcesswithMicroarrays,andSpotChapter4,page23.RNAAnalysisNotebookTableofContentsWorkingwithRNA2AnalyzingRNAwithEstablishanRNase-FreeEnvironmentMicroarrays25MaintainRNase-FreeConditionsinReactionsOverviewGelAnalysisofRNAProbeProductionTargetProduction

3、PurifyingRNAandmRNA7PurificationRequirementsMakingRNAinvitro30Large-scaleTotalRNAIsolationOverviewMiniprep-scaleTotalRNAIsolationRiboProbe®invitroTranscriptionSystems96-WellTotalRNAIsolationRiboMAXTMLargeScaleRNAProductionSystemsAutomatedTotalRNAIsolationEukaryoticinv

4、itroTranscriptionPoly(A)+RNAIsolationPrecipitationofRNASamplesDownstreamApplicationsUsingRNASilencingRNAinvivo(RNAi)35AmplifyingRNAwithRT-PCR16RNAiPhenomenonMammalianRNAiRNAiTargetSelectionfortheOverviewAccessQuickTMandAccessRT-PCRSystemssiLentGene™SystemTheImProm-II

5、TMRTSystemandReverseDownstreamPrimerSequenceforusewiththesiLentGeneSystemTranscriptionSystemLongdouble-strandedRNAforReverseTranscriptasesnon-mammalianRNAiTipsandTechniqueswww.promega.com•techserv@promega.comWorkingwithRNAWearglovesandusedisposable,EstablishanRNase-F

6、reeEnvironmentsterileplasticware!WhenworkingwithRNA,caremustbetakentocreatearibonuclease-freeenvironment.RibonucleasesorRNasesareeverywhere!RNasesareverystableanddifficulttoinactivate.Toensuresuccess,itisimportanttomaintainaRNase-freeenvironmentstartingwithRNApurificat

7、ionandcontinuingthroughanalysis.BelowaresometipsandtechniquestorememberwhenworkingwithRNA:1.ThemostcommonsourcesofRNasethetargetorprobeRNAoccurs,itmaybecontaminationarehands(skin)andbacteriaornecessarytotestthewatersourceforRNasemoldthatmaybepresentonairbornedustpart

8、iclesactivitybyincubatinganRNAsamplewiththeorlaboratoryglassware.Topreventcontaminationwaterandcheckingfordegradationbygelfromthese