Gene expression analysis using single molecule detection.
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.
Korn
Kerstin
K
Gnothis AB, Electrum 212, SE-164 40 Kista, Sweden.
Gardellin
Paola
P
Liao
Bohao
B
Amacker
Mario
M
Bergström
Asa
A
Björkman
Henrik
H
Camacho
Agnès
A
Dörhöfer
Sabine
S
Dörre
Klaus
K
Enström
Johanna
J
Ericson
Thomas
T
Favez
Tatiana
T
Gösch
Michael
M
Honegger
Adrian
A
Jaccoud
Sandra
S
Lapczyna
Markus
M
Litborn
Erik
E
Thyberg
Per
P
Winter
Holger
H
Rigler
Rudolf
R
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
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