title: Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization.
abstract: The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.
authors.affiliation: Department of Neurology, Mount Sinai School of Medicine, One Gustave L. Levy Place, NY 10029, USA.
authors.forename: PokMan
authors.initials: P
authors.lastname: Chan
authors.forename: Tony
authors.initials: T
authors.lastname: Yuen
authors.forename: Frederique
authors.initials: F
authors.lastname: Ruf
authors.forename: Javier
authors.initials: J
authors.lastname: Gonzalez-Maeso
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Sealfon
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008562:Membrane Glycoproteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D051379:Mice; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000302:isolation & purification; D038761:Photobleaching; D045663:Quantum Dots; D012333:RNA, Messenger / Q000032:analysis*; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
chemical_list: D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C493453:Slc18a2 protein, mouse; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins