Chapter 5 Detection of mRNA Species by Flow Cytometry

Publisher Summary This chapter describes two methods for revealing poly(A) + RNA that are particularly suited to flow cytometric analysis. Various attempts to detect rRNA or mRNA by flow cytometry (FCM) have been described using either biotinylated probes and fluorescent in situ hybridization (FISH) or primed in situ labeling (PRINS). Such methods are derived from microscopic and morphologic methodologies. Analysis of FISH and FCM has been hampered by two main problems. First, although the photomultipliers used in FCM can detect low fluorescence intensities, they measure the entire fluorescence signal for each cell and cannot resolve the pattern of fluorescence within the cell. Target-specific fluorescence is not therefore readily discriminated from background fluorescence. The second problem is the “sponge-like” behavior of fixed permeabilized cells in suspension, which tend to trap macromolecules in their cytoplasm. This considerably increases the nonspecific fluorescence when fluorescent proteins such as avidin or antibodies are used to reveal the probes. In view of these drawbacks, a more specific labeling method is required for flow cytometric analysis of FISH, which takes both the characteristics of the detectors and the properties of the cell suspensions into account.