Quantitative measurement of transcription dynamics in living cells

In a wide range of organisms the kinetics of transcription have been found to be noisy, with "bursts" or "pulses" of transcription interspersed with irregular periods of inactivity. The in vivo analysis of transcription dynamics can be most directly monitored using RNA stem loop motifs derived from MS2 and other bacteriophages. Here we describe the implementation of the MS2 RNA detection system and the steps required for precise measurement of transcription dynamics in highly motile cells. Automated image processing techniques are used to track large numbers of cells and measure transcription in a systematic and unbiased manner. We discuss popular methods for automatic image segmentation and frame-to-frame tracking of cells, and the considerations required to make measurements as quantitatively as possible.