Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization.

Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signaling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here, we introduce qFIT (quantitative forced intercalation) probes that allow the detection both of probe-target complexes and of unbound probes on separate, independent channels. A surrogate nucleobase based on thiazole orange (TO) probes the hybridization status. The second channel involves a nonresponsive near-IR dye, which serves as a reporter of concentration. We show that the undesirable perturbation of the hybridization reporter TO is avoided when the near-IR dye Cy7 is connected by means of short triazole linkages in an ≥18 nucleotides distance. We used the qFIT probes to localize and quantify oskar mRNA in fixed egg chambers of wild-type and mutant Drosophila melanogaster by wash-free fluorescence in situ hybridization. The measurements revealed a relative 400-fold enrichment of oskar within a 3000 μm large volume at the posterior pole of stage 8-9 oocytes, which peaked at a remarkably high 1.8 μM local concentration inside 0.075 μm volume units. We discuss detection limits and show that the number of oskar mRNA molecules per oocyte is independent of the oocyte size, which suggests that the final levels are attained already during the onset of oskar localization at stage 8.