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mRNA turnover plays an important role in the regulation of post-transcriptional gene expression. While many protein factors involved in mRNA degradation have been identified, we still lack a basic understanding of the principles that regulate the spatiotemporal dynamics of mRNA turnover within single cells. To overcome this limitation, we have developed the TREAT biosensor, which allows for discrimination of intact reporter transcripts and stabilized decay intermediates using single RNA imaging. Here, we present an image analysis pipeline that performs semiautomated detection and tracking of individual mRNA particles. It colocalizes tracks and applies the colocalization information to quantify the number of intact transcripts and degradation intermediates. Based on the analysis of control data, the workflow further determines detection efficiencies and uses them to correct RNA particle numbers.

Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure.