Allele-specific detection of single mRNA molecules in situ.
We describe a method for fluorescence in situ identification of individual mRNA molecules, allowing quantitative and accurate measurement, in single cells, of allele-specific transcripts that differ by only a few nucleotides. By using a combination of allele-specific and non-allele-specific probe libraries, we achieve >95% detection accuracy. We investigate the allele-specific stochastic expression of Nanog, which encodes a pluripotency factor, in murine embryonic stem cells.
Harvard University Graduate Biophysics Program, Harvard Medical School, Boston, Massachusetts, USA.
Clinton H
CH
Hansen
Alexander
A
van Oudenaarden
D000465:Algorithms; D000483:Alleles; D000818:Animals; D002232:Carbocyanines / Q000032:analysis; D002678:Chimera; D053595:Embryonic Stem Cells / Q000502:physiology; D005456:Fluorescent Dyes / Q000032:analysis; D018398:Homeodomain Proteins / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D051379:Mice; D008815:Mice, Inbred Strains; D015335:Molecular Probes; D000071317:Nanog Homeobox Protein; D009930:Organic Chemicals / Q000032:analysis; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000032:analysis*; D059010:Single-Cell Analysis / Q000379:methods*; D013269:Stochastic Processes
C417664:Alexa594; D002232:Carbocyanines; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins; D015335:Molecular Probes; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D009930:Organic Chemicals; D012333:RNA, Messenger; C085321:cyanine dye 5