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 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.
Camacho
Agnès
A
Gnothis SA, PSE-B, EPFL, CH-1015 Lausanne, Switzerland.
Korn
Kerstin
K
Damond
Martine
M
Cajot
Jean-François
JF
Litborn
Erik
E
Liao
Bohao
B
Thyberg
Per
P
Winter
Holger
H
Honegger
Adrian
A
Gardellin
Paola
P
Rigler
Rudolf
R
D002135:Calcium-Binding Proteins; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003598:Cytoskeletal Proteins / Q000032:analysis; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005456:Fluorescent Dyes; D015870:Gene Expression; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D008562:Membrane Glycoproteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis; D010735:Phosphoglycerate Kinase / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D012235:Rhodamines; D012680:Sensitivity and Specificity; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D050857:Synaptotagmins; D013997:Time Factors
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; C482331:DNMBP protein, human; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D012235:Rhodamines; D012996:Solutions; D050857:Synaptotagmins; D010735:Phosphoglycerate Kinase