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Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.