Fluorescence in situ hybridization analysis of transcript dynamics in cells

Since the first description of in situ hybridization in 1969 the technique has advanced to allow sensitive detection of DNA and mRNA molecules at the cellullar and subcellular levels. In particular fluorescence in situ hybrid ization (FISH) has become a frequently used tool in basic and applied biome dical research since detection is sensitive and allows discrimination of mu ltiple targets in the same sample. By using RNA-FISH we have been able to d etect primary transcripts of the human embryonic, fetal, and adult globins in erythroid cells to study the competitive transcription mechanism or vari egated expression patterns of the human,beta -globin locus. We have correla ted such expression patterns with other parameters such as cell type, cell cycle, replication, and stage of differentiation by simultaneous detection of, e.g" incorporated BrdUTPs, proteins (e.g., cyclins A and E, PCNA, histo ries), and globin (primary) transcripts and/or locus integration sites. Thu s a combination of FISH and immunofluorescence methods allow the visualizat ion of different processes taking place in the nucleus relative to each oth er in terms of three-dimensional space and structure and time (development, cell cycle). (C) 2001 Academic Press.