NormaCurve A SuperCurve-Based Method That Simultaneously Quantifies and Normalizes Reverse Phase Protein Array Data 英文参考文献.docVIP

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NormaCurve A SuperCurve-Based Method That Simultaneously Quantifies and Normalizes Reverse Phase Protein Array Data 英文参考文献.doc

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NormaCurve A SuperCurve-Based Method That Simultaneously Quantifies and Normalizes Reverse Phase Protein Array Data 英文参考文献

NormaCurve:ASuperCurve-BasedMethodThat SimultaneouslyQuantifiesandNormalizesReverse PhaseProteinArrayData SylvieTroncale1,3,4,Aure′lieBarbet2,LamineCoulibaly2,EmilieHenry2,BeileiHe2,EmmanuelBarillot1, ThierryDubois2,PhilippeHupe′1,3,4,5,LeannedeKoning2* 1InstitutCurie,Paris,France,2DepartmentofTranslationalResearch,InstitutCurie,Paris,France,3INSERM,U900,Paris,France,4MinesParisTech,Fontainebleau,France, 5CNRSUMR144,Paris,France Abstract Motivation:Reversephaseproteinarray(RPPA)isapowerfuldot-blottechnologythatallowsstudyingproteinexpression levelsaswellaspost-translationalmodificationsinalargenumberofsamplessimultaneously.Yet,correctinterpretationof RPPAdatahasremainedamajorchallengeforitsbroad-scaleapplicationanditstranslationintoclinicalresearch.Satisfying quantification tools are available to assess a relative protein expression level from a serial dilution curve. However, appropriatetoolsallowingthenormalizationofthedataforexternalsourcesofvariationarecurrentlymissing. Results:Hereweproposeanewmethod,calledNormaCurve,thatallowssimultaneousquantificationandnormalizationof RPPAdata.Forthis,wemodifiedthequantificationmethodSuperCurveinordertoincludenormalizationfor(i)background fluorescence,(ii)variationinthetotalamountofspottedproteinand(iii)spatialbiasonthearrays.Usingaspike-indesign withapurifiedprotein,wetestthecapacityofdifferentmodelstoproperlyestimatenormalizedrelativeexpressionlevels. Thebestperformingmodel,NormaCurve,takesintoaccountanegativecontrolarraywithoutprimaryantibody,anarray stainedwithatotalproteinstainandspatialcovariates.Weshowthatthisnormalizationisreproducibleandwediscussthe numberofserialdilutionsandthenumberofreplicatesthatarerequiredtoobtainrobustdata.Wethusprovideaready-to- use method for reliable and reproducible normalization of RPPA data, which should facilitate the interpretation and the developmentofthispromisingtechnology. Availability: The raw data, the scripts and the NormaCurve package are available at the following web site: