Transcript quantification based on chemical labeling of RNA associated with fluorescent detection

A general method for RNA measurement, based on chemical labeling of RNA wit h digoxigenin (without retrotranscription), has been established. Labeled R NA is hybridized with nylon membranes containing spot blots of MR-amplified gene fragments and the fluorescence detection is mediated via specific ant idigoxigenin antibody coupled to alkaline phosphatase. The method was optim ized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene express ion. When the quantity of cellular RNA used in this method is maintained co nstant 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 ext ended to macroarrays blotted automatically and the validity of the method w as tested by comparison with expression data obtained by Northern blotting. (C) 2001 Academic Press.