Visualization of single RNA transcripts in situ.
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.
Femino
A M
AM
Department of Anatomy and Structural Biology and Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Fay
F S
FS
Fogarty
K
K
Singer
R H
RH
D000199:Actins / Q000235:genetics; D000818:Animals; D002460:Cell Line; D016650:Fluorescein-5-isothiocyanate; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D015345:Oligonucleotide Probes; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic
D000199:Actins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D016650:Fluorescein-5-isothiocyanate