简介：ThelocalCa^2+releasefromtheheterogeneouslydistributedendoplasmicreticulum(ER)calciumstorehasacriticalroleincalciumhomeostasisandcellularfunction.However;singlefluorescentproteinbasedERcalciumprobesexperiencechallengesinquantifyingtheERcalciumstoreindifferinglivecells,andintensity-basedmeasurementsmakeitdifficulttodetectlocalcalciummicrodomainsintheER.Here,wedevelopedageneticallyencodedratiometricERcalciumindicator[GCEPIA1-SNAPer]thatcandetectthereal-timeERcalciumstoreandlocalcalciummicrodomainsinlivecells.GCEPIA1-SNAPerwaslocatedinthelumenoftheERandshowedalinear;reversibleandrapidresponsetochangesintheERcalciumstore.TheGCEPIA1-SNAPerprobeeffectivelymonitoredthedepletionoftheERcalciumstorebyTGorstarvationtreatment,andthrough让suseweidentifiedheterogeneouslydistributedcalciummicrodomainsintheERwhichwerecorrelatedw让hthedistributionofSTIM1clustersuponERcalciumstoredepletion.Lastly,GCEPIA1-SNAPercanbeusedtodetecttheERcalciumstorebyhigh-throughputflowcytometryandconferstheabilitytostudythefunctionofcalciummicrodomainsoftheER.

简介：Anon-radioisotopic,quantitativeTRAP-basedtelomeraseactivityassaywasestablishedmainlybyusingSYBRGreen-Istaininginsteadofradioisotope.Comparingwithconventionalradioisotopebasedmethod,itwasbetterinreproducibilityandaccuracy.Usingthismethod,wefoundtelomeraseactivitieswereabsentinnormalhumanlivercells,whiledetectedinalloffourhumanhepatomacelllines(BEL-7404,SMMC-7721,QGY-07903andHCCM)withoutsignificantdifferences.

简介：Quantitativegeneexpressionanalysisplaysanimportantroleinidentifyingdifferentiallyexpressedgenesinvariouspathologicalstates,geneexpressionregulationandco-regulation,sheddinglightongenefunctions.Althoughmicroarrayiswidelyusedasapowerfultoolinthisregard,itissuboptimalquantitativelyandunabletodetectunknowngenevariants.Herewedemonstratedeffectivedetectionofdifferentialexpressionandco-regulationofcertaingenesbyexpressedsequencetaganalysisusingaselectedsubsetofcDNAlibraries.Wediscussedtheissuesofsequencingdepthandlibrarypreparation,andproposethatincreasedsequencingdepthandimprovedpreparationproceduresmayallowdetectionofmanyexpressionfeaturesforlessabundantgenevariants.Withthereductionofsequencingcostandtheemergingofnewgenerationsequencingtechnology,in-depthsequencingofcDNApoolsorlibrariesmayrepresentabetterandpowerfultoolingeneexpressionprofilingandcancerbiomarkerdetection.Wealsoproposeusingsequence-specificsubtractiontoremovehundredsofthemostabundanthousekeepinggenestoincreasesequencingdepthwithoutaffectingrelativeexpressionratioofothergenes,astranscriptsfromasfewas300mostabundantlyexpressedgenesconstituteabout20%ofthetotaltranscriptome.In-depthsequencingalsorepresentsauniqueadvantageofdetectingunknownformsoftranscripts,suchasalternativesplicingvariants,fusiongenes,andregulatoryRNAs,aswellasdetectingmutationsandpolymorphismsthatmayplayimportantrolesindiseasepathogenesis.

简介：Theplasmamembrane(PM)isacomplexenvironmentconsistingof>700speciesoflipidsandmanydifferenttypesofmembrane-associatingproteins.Theselipidsandmembraneproteinsaredistributedheterogeneouslyintonanometer-sizeddomains,callednanoclusters.ThelateralspatialsegregationinthePMgivesrisetodifferentcurvatureandlipidcomposition,whichdeterminestheefficiencyofeffectorbindingandsignaltransmission.Here,wedescribeanelectronmicroscopy(EM)-spatialmappingtechniquetoquantifytheextentofnanoclustersformationinthePM.Thenano-assembliesinthePMarequantifiedviaexpressingtheGFP-taggedproteinsorlipid-bindingdomainsinthecells,whicharethenimmunolabeledwiththegoldnanoparticlespre-coupledtotheanti-GFPantibody.ThegoldnanoparticlesarevisualizedviathetransmissionEMathighmagnification.ThestatisticalanalysisoftheRipley'sK-functioncalculatesthespatialdistributionofthegoldnanoparticles.Importantspatialparameters,suchastheextentofnanoclustering,theclusteredfraction,thenumberofproteinspercluster,theoptimalsizeofananocluster,andthenumberofproteinslocalizedtothePM,canbecalculated.Furtherdetailedaggregationpattern,suchasthepopulationsofmonomers,dimers,trimers,andhigherorderedoligomers,canalsobeextractedfromthespatialanalysis.TheEM-bivariateanalysisquantifiestheextentofco-localizationbetweentwodifferentcomponentsinthePMandprovideskeyinformationontheprotein-proteinandtheprotein-lipidinteractionsoveralong-distancescalefrom8to240nm.