title: Quantitative measurement of transcription dynamics in living cells.
abstract: 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.
authors: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom.
authors: Adam M
authors: AM
authors: Corrigan
authors: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom.
authors: Jonathan R
authors: JR
authors: Chubb
mesh_terms: D000465:Algorithms; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival; D003584:Cytological Techniques / Q000379:methods*; D004023:Dictyostelium / Q000166:cytology* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017909:Levivirus / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D036022:Capsid Proteins; D049452:Green Fluorescent Proteins
keywords: Automated image analysis; Automatic image segmentation; Dictyostelium; Gene expression noise; High content imaging; MS2; RNA FISH; Single cell transcriptomics; Stochastic gene expression; Transcriptional bursting