Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes☆

摘要

AsymmetricRNAprobes,whichcontainonlythemRNAcodingstrand,providealargeincreaseinhybridizationefficiencyinsituoverthatobservedwitheithersymmetric(bothstrandsrepresented)RNAorDNAprobes.AsymmetricRNAprobesaresynthesizedinvitrobytranscriptionfromrecombinantsformedbetweensequencesencodingseaurchinmRNAsandthetranscriptionvectorR7?”7.UsingaproberepresentingearlyvarianthistonemRNAsequenceswehavecharacterizedhybridizationtosectionsofseaurchinembryoswithrespecttothermalstabilityofthehybridsformed,optimumtemperature,effectofsequencedivergenceonhybridthermalstability,anddependenceofthehybridizationsignalsonprobeconcentrationandhybridizationtime.EstimatesfromtheobservedsignalsindicatethatalargefractionoftargetRNAsisbothretainedinsectionsandhybridizedwithprobeatsaturation.Coupledwithmeasurementsofnonspecificbackgroundbindingofheterologousprobes,thesedataindicatethatthemethodhassufficientsensitivitytodetectmanymoderatelyabundantmRNAs(20a€“75moleculespercellinthe1500-cellpluteus).InsituhybridizationstoembryosatdifferentdevelopmentalstagesshowthatwhilehistonemRNAsareuniformlydistributedincleavingembryos,differentcelllineagesofolderembryosshowlargedifferencesinaccumulationofthesemRNAs.