Direct quantification of mRNA expression levels using single molecule detection

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 t...

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Bibliographic Details
Published in:Journal of biotechnology 2004, Vol.107 (2), p.107-114
Main Authors: Camacho, Agnès, Korn, Kerstin, Damond, Martine, Cajot, Jean-François, Litborn, Erik, Liao, Bohao, Thyberg, Per, Winter, Holger, Honegger, Adrian, Gardellin, Paola, Rigler, Rudolf
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Calcium-Binding Proteins
Calibration
Chromatography, High Pressure Liquid
Cytoskeletal Proteins - analysis
DNA, Complementary - chemical synthesis
DNA, Complementary - chemistry
DNA, Complementary - genetics
DNA, Complementary - isolation & purification
DNA, Complementary - metabolism
Fluorescence correlation spectroscopy
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Gene Expression
Gene expression analysis
HL-60 Cells
Humans
K562 Cells
Medicin och hälsovetenskap
Membrane Glycoproteins - analysis
mRNA analysis
Nerve Tissue Proteins - analysis
Phosphoglycerate Kinase - analysis
Reproducibility of Results
Rhodamines
RNA, Messenger - analysis
RNA, Messenger - genetics
Sensitivity and Specificity
Single molecule detection
Solutions
Spectrometry, Fluorescence - methods
Spectrophotometry, Ultraviolet
Synaptotagmins
Time Factors
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Description
Summary: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.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2003.10.003