Gene expression analysis using single molecule detection

• Published in: Nucleic acids research Vol. 31; no. 16; p. e89
• Main Authors: Korn, Kerstin, Gardellin, Paola, Liao, Bohao, Amacker, Mario, Bergström, Åsa, Björkman, Henrik, Camacho, Agnès, Dörhöfer, Sabine, Dörre, Klaus, Enström, Johanna, Ericson, Thomas, Favez, Tatiana, Gösch, Michael, Honegger, Adrian, Jaccoud, Sandra, Lapczyna, Markus, Litborn, Erik, Thyberg, Per, Winter, Holger, Rigler, Rudolf
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.08.2003; Oxford Publishing Limited (England)
• Subjects: Actins - genetics; Algorithms; Carbocyanines - chemistry; DNA, Complementary - chemistry; DNA, Complementary - genetics; DNA, Complementary - metabolism; DNA-Binding Proteins - genetics; Fluorescent Dyes - chemistry; Gene Expression; HL-60 Cells; Humans; K562 Cells; Medicin och hälsovetenskap; NAR Methods Online; NF-kappa B - genetics; Oligonucleotide Probes - chemistry; Oligonucleotide Probes - genetics; Peptide Elongation Factor 1 - genetics; Phosphoglycerate Kinase - genetics; rab1 GTP-Binding Proteins - genetics; Rhodamines - chemistry; Ribosomal Proteins - genetics; Sensitivity and Specificity; Spectrometry, Fluorescence - instrumentation; Spectrometry, Fluorescence - methods; Transcription Factor RelA; Tubulin - genetics
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gng089

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.