RESUMO
Determination of the gene expression by direct quantification of mRNA is becoming increasingly important in basic, pharmaceutical and clinical research. We present a novel approach for gene quantification based on direct hybridization of gene-specific probes to target mRNA sequences in solution at temperatures ensuring absolute specificity of the probe-target complex. No enzymatic steps like reverse transcription or amplification by PCR are involved within the quantification process. In order to increase specificity as well as sensitivity, two probes emitting fluorescence light in different colors are used for our homogeneous assay using fluorescence cross-correlation. We relate to the single molecule sensitivity of excitation and detection in confocal cavities avoiding the amplification of the detected signal. The analysis of the expression level of high, medium and low abundant genes is described in two different cell lines, whereby the genes are quantified in absolute numbers.
Assuntos
Proteínas de Ligação ao Cálcio , DNA Complementar/metabolismo , Expressão Gênica , RNA Mensageiro/análise , RNA Mensageiro/genética , Espectrometria de Fluorescência/métodos , Calibragem , Cromatografia Líquida de Alta Pressão , Proteínas do Citoesqueleto/análise , DNA Complementar/síntese química , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Corantes Fluorescentes , Células HL-60 , Humanos , Células K562 , Glicoproteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Fosfoglicerato Quinase/análise , Reprodutibilidade dos Testes , Rodaminas , Sensibilidade e Especificidade , Soluções , Espectrofotometria Ultravioleta , Sinaptotagminas , Fatores de TempoRESUMO
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.
