Ambion rnasealert lab test kit

Ambion RNaseAlert Lab Test kit (Thermo Fisher Scientific, #AM1964) was used to detect RNase enzymatic activity according to the manufacturer’s instruction. Briefly, 5 μl of 10-fold RNaseAlert buffer was added to a tube containing the fluorescent substrate, and then mixed with a total of 45 μl of the tested CM with 1/1000 dilution by RNase-free water to reduce background RNase activity. The mixture was sequentially placed on a well of a 96-well plate. The real-time fluorescence data were collected at 1 min intervals for 21 min using a BioTek Synergy™ Neo multi-mode reader (BioTek Instruments).

Different concentration of Si-OH or Si-NH2 NPs were incubated with RNase in PBS pH 7.4 for 1 h. In order to discard the excess of unbound protein, the mixture was filtered through a Vivaspin20 ultrafilter (cutoff = 30000 Da, Vivascience-Sartorius, Germany) and washed three times with PBS pH 7.4. Subsequently, RNase activity was assessed using Ambion® RNaseAlert® Lab Test kit (Thermo Fisher Scientific), according to the manufacturer’s instruction. The kit contains a fluorescent substrate that emits a green fluorescence if it is cleaved by RNase, the fluorescence can be visually detected by short-wave UV illumination or measured in a fluorometer. Solutions with active RNase produce a green glow in the assay, whereas solutions without RNase activity do not fluoresce. Following staining, samples were analyzed using a fluorescence microplate reader (Tecan Infinite® 200 PRO). Readings were done in quadruplicates.