natureprotocols单细胞定量PCR

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1、nature

2、methodsQuantitativeanalysisofgeneexpressioninasinglecellbyqPCRKiyomiTaniguchi,TomoharuKajiyama&HidekiKambaraSupplementaryfiguresandtext:SupplementaryFigure1RTefficienciesoffourprobesand3′biasincDNAsynthesis.SupplementaryFigure2StructureofmodelRNAa

3、ndtargetregionforqPCR.SupplementaryFigure3Selectionofoptimumreversetranscriptase.SupplementaryFigure4EvaluationofreproducibilityinreuseofstandardssDNAtemplates.SupplementaryTable1Primersequences.SupplementaryTable2RTreactionconditions.SupplementaryTable3

4、ImmobilizationefficiencyofdsDNAonbeads.SupplementaryTable4StandarddeviationinqPCR.SupplementaryTable5Geneexpressionlevelsoffourgenesinsinglecells.SupplementaryTable6MeasuredamountsofcDNAandstandarddeviations.SupplementaryProtocolSupplementaryMethodsNatur

5、eMethods:doi:10.1038/nmeth.1338SupplementaryFigure1.RTefficienciesoffourprobesand3’biasincDNAsynthesisab1000TBP10001,000SDHA800B2M800800600600600400400400200200200NumberofcDNAmolecules0NumberofcDNAmolecules00(1)(2)(3)(4)003003006006009009001,20012001,500

6、15001,80018002,1002100Distancefrom3’endtotargetregion(bases)Probesimmobilizedonbeads(a)RTefficiencieswithfourtypesofprobes(1)oligo(dT)30,(2)gene-specific,(3)oligo(dT)VN,and(4)LNAforamodelRNA(SDHA,103molecules)wereroughlythe253moleculesofmodelRNA(same(mea

7、ns.d.,n=3).(b)With10TBP,SDHA,andB2M),the3’biaswasevaluatedbycarryingoutqPCRatdifferenttargetregionofthecDNAspecies(means.d.,n=3).TheestimatednumberofcopiesdecreasedwiththedistancebetweenthePCRportionsand3’terminiinaregionover500basesfromthetermini.Natu

8、reMethods:doi:10.1038/nmeth.1338SupplementaryFigure2.StructureofmodelRNAandtargetregionforqPCRTBPRegion2TBPRegion1(301－462bases)(0－113bases)a5’345678AAAAAA3’844basesSDHARegion1(52－143bases)SDHARegion4SDHARegion3SDHARegion2(2000－2110bases)(1100－1184bases)

9、(179－302bases)b5’123456789101112131415AAAAAA3’2161basesB2MRegion2B2MRegion1(374－494bases)(115－226bases)c5’234AAAAAA3’838basesStructureofmodelRNA(a:TBP,b:SDHA,andc:B2M)andq-PCRprimers(redarrows)andprobes(bluebars)atdifferen