Fluorescence in situ hybridization analysis of transcript dynamics in cells.

ABSTRACT

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 cellular and subcellular levels. In particular fluorescence in situ hybridization (FISH) has become a frequently used tool in basic and applied biomedical research since detection is sensitive and allows discrimination of multiple targets in the same sample. By using RNA-FISH we have been able to detect primary transcripts of the human embryonic, fetal, and adult globins in erythroid cells to study the competitive transcription mechanism or variegated expression patterns of the human beta-globin locus. We have correlated 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, histones), and globin (primary) transcripts and/or locus integration sites. Thus a combination of FISH and immunofluorescence methods allow the visualization of different processes taking place in the nucleus relative to each other in terms of three-dimensional space and structure and time (development, cell cycle).