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1、METHODSINMOLECULARBIOLOGYMETHODSINMOLECULARBIOLOGYTMTMVolume258GeneExpressionGeneExpressionProfilingProfilingMethodsandProtocolsMethodsandProtocolsEditedbyEditedbyRichardA.ShimketsRichardA.ShimketsTechnicalConsiderations11TechnicalConsiderationsinQuantitatingG
2、eneExpressionRichardA.Shimkets1.IntroductionScientistsroutinelylectureandwriteaboutgeneexpressionandtheabun-danceoftranscripts,butinreality,theyextrapolatethisinformationfromavari-etyofmeasurementsthatdifferenttechnologiesmayprovide.Indeed,therearemanyreasonst
3、hatapplyingdifferenttechnologiestotranscriptabundancemaygivedifferentresults.Thismayresultfromanincompleteunderstandingofthegeneinquestionorfromshortcomingsintheapplicationsofthetechnologies.Thefirstkeyfactortoappreciateinmeasuringgeneexpressionisthewaythatgen
4、esareorganizedandhowthisinfluencesthetranscriptsinacell.Figure1depictssomeofthescenariosthathavebeendeterminedfromsequenceanalysesofthehumangenome.Mostgenesarecomposedofmultipleexonstranscribedwithintronsequencesandthensplicedtogether.Somegenesexistentirelybet
5、weentheexonsofothergenes,eitherintheforwardorreverseorientation.Thisposesaproblembecauseitispossibletorecoverafragmentorclonethatcouldbelongtomultiplegenes,bederivedfromanunsplicedtranscript,orbetheresultofgenomicDNAcontaminatingtheRNApreparation.Alloftheseeve
6、ntscancreateconfusingandconfoundingresults.Additionally,thegenedup-licationeventsthathaveoccurredinorganismsthataremorecomplexhaveledtotheexistenceofcloselyrelatedgenefamiliesthatcoincidentallymaylieneareachotherinthegenome.Inaddition,althoughthereareprobablyl
7、essthan50,000humangenes,theexonswithinthosegenescanbesplicedtogetherinavarietyofways,withsomegenesdocumentedtoproducemorethan100differenttran-scripts(1).From:MethodsinMolecularBiology,Vol.258:GeneExpressionProfiling:MethodsandProtocolsEditedby:R.A.Shimkets©Hum
8、anaPressInc.,Totowa,NJ12ShimketsFig.1.Typicalgeneexonstructure.Therefore,theremaybeseveralhundredthousanddistincttranscripts,withpotentiallymanycommonsequences.Genebiologyisevenmor
