title: Gene expression analysis using single molecule detection.
abstract: Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.
authors.affiliation: Gnothis AB, Electrum 212, SE-164 40 Kista, Sweden.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Korn
authors.forename: Paola
authors.initials: P
authors.lastname: Gardellin
authors.forename: Bohao
authors.initials: B
authors.lastname: Liao
authors.forename: Mario
authors.initials: M
authors.lastname: Amacker
authors.forename: Asa
authors.initials: A
authors.lastname: Bergström
authors.forename: Henrik
authors.initials: H
authors.lastname: Björkman
authors.forename: Agnès
authors.initials: A
authors.lastname: Camacho
authors.forename: Sabine
authors.initials: S
authors.lastname: Dörhöfer
authors.forename: Klaus
authors.initials: K
authors.lastname: Dörre
authors.forename: Johanna
authors.initials: J
authors.lastname: Enström
authors.forename: Thomas
authors.initials: T
authors.lastname: Ericson
authors.forename: Tatiana
authors.initials: T
authors.lastname: Favez
authors.forename: Michael
authors.initials: M
authors.lastname: Gösch
authors.forename: Adrian
authors.initials: A
authors.lastname: Honegger
authors.forename: Sandra
authors.initials: S
authors.lastname: Jaccoud
authors.forename: Markus
authors.initials: M
authors.lastname: Lapczyna
authors.forename: Erik
authors.initials: E
authors.lastname: Litborn
authors.forename: Per
authors.initials: P
authors.lastname: Thyberg
authors.forename: Holger
authors.initials: H
authors.lastname: Winter
authors.forename: Rudolf
authors.initials: R
authors.lastname: Rigler
mesh_terms: D000199:Actins / Q000235:genetics; D000465:Algorithms; D002232:Carbocyanines / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D016328:NF-kappa B / Q000235:genetics; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D012235:Rhodamines / Q000737:chemistry; D012269:Ribosomal Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D051996:Transcription Factor RelA; D014404:Tubulin / Q000235:genetics; D020761:rab1 GTP-Binding Proteins / Q000235:genetics
chemical_list: D000199:Actins; D002232:Carbocyanines; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D020648:Peptide Elongation Factor 1; D012235:Rhodamines; D012269:Ribosomal Proteins; D051996:Transcription Factor RelA; D014404:Tubulin; C085321:cyanine dye 5; C040343:ribosomal protein S19; D010735:Phosphoglycerate Kinase; D020761:rab1 GTP-Binding Proteins