2.3. RNA Labeling and Fragmentation
The PlatinumBright Infrared Labeling Kit from KREATECH (Amsterdam, Netherlands) was used to label 1.5 µg RNA of field sample using 2 µL ULS dye and 2 µL 10× labeling solution in a total volume of 20 µL. Samples were labeled by incubation for 30 min at 85 °C. After incubation, samples were placed on ice and spun down and then purified with KREApure columns (KREATECH) according to the manufacturer’s instructions. Concentration and incorporation of the dye was measured by a NanoVue (GE Healthcare). The DoL (degree of labeling) was calculated and was between 1.9 and 2.2% (Table 1). RNA was fragmented by adding 1/10 volume of RNA fragmentation buffer (100 mM ZnCl2 in 100 mM Tris-HCL, pH 7.0) and an incubation of 15 min at 70 °C. The reaction was stopped with the addition of 1/10 volume of 0.5 M EDTA (pH 8.0) and the samples were placed on ice. The RNA was fragmented to reduce the effect of the secondary structure on the accessibility of the probe. Despite this fragmentation, we still have heterogeneous probe sensitivity, which reflects the influence of the secondary structure and we can only partially overcome this by fragmenting the RNA to remove the strongest secondary structure formations.