Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.
A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression. When the quantity of cellular RNA used in this method is maintained constant and the amount of RNA in the cell determined, the true intracellular transcript concentrations can be determined, rather than simple abundance of a messenger in RNA population. This RNA quantification technique was extended to macroarrays blotted automatically and the validity of the method was tested by comparison with expression data obtained by Northern blotting.
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Centre de Bioingénierie Gilbert Durand, UMR 5504 INSA/CNRS and UMR 792 INSA/INRA, Institut National des Sciences Appliquées, 135 Avenue de Rangueil, Toulouse Cedex 4, 31077, France.
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D015152:Blotting, Northern; D003013:Clostridium / Q000235:genetics* / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004076:Digoxigenin / Q000378:metabolism; D005453:Fluorescence; D020869:Gene Expression Profiling; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis* / Q000378:metabolism
D008567:Membranes, Artificial; D012329:RNA, Bacterial; D003850:Deoxyribonuclease I; D004076:Digoxigenin