Quantitative Image Analysis of Single-Molecule mRNA Dynamics in Living Cells
Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm. In order to measure the fluorescence associated with individual RNA molecules over time, we developed a semi-automated quantitative image analysis tool termed STaQTool. We describe in detail the implementation and application of the STaQTool software package, which is a generic tool able to process large 4D datasets allowing quantitative studies of different steps in gene expression.
Key wordsLive-cell imaging Single-molecule Fluorescence microscopy Fluorescence quantification RNA splicing
We gratefully acknowledge Tomas Kirchhausen and members of the Kirchhausen lab for advice and insightful discussion. This work was supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/118180/2010; FCT-ANR/BIM-ONC/0009/2013), and the Harvard Medical School-Portugal Program in Translational Research and Information.
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