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Fluorescence
in situ hybridization (FISH) and digital imaging microscopy were
modified to allow detection of single RNA molecules. Oligodeoxynucleotide
probes were synthesized with five fluorochromes per molecule, and
the light emitted by a single probe was calibrated. Points of light
in exhaustively deconvolved images of hybridized cells gave fluorescent
intensities and distances between probes consistent with single
messenger RNA molecules. Analysis of beta-actin transcription sites
after serum induction revealed synchronous and cyclical transcription
from single genes. The rates of transcription initiation and termination
and messenger RNA processing could be determined by positioning
probes along the transcription unit. This approach extends the power
of FISH to yield quantitative molecular information on a single
cell. |
