Micrococcal nuclease enzyme mnase

The potential environmental degradation of dsRNA was investigated by exposure of naked-Bc VPS51+DCTN+SAC1-dsRNA (200 ng) and AV-Bc-VDS-dsRNA (200 ng/2.5 μg) to Micrococcal Nuclease enzyme (MNase) (Thermo Fisher) treatment in four replicate experiments. Samples were treated with 0.2 U μL⁻¹ MNase for 10 min at 37 °C, and dsRNAs were released using 1% Triton X-100. All samples were visualized on a 2% agarose gel. The persistence of sprayed naked-Bc-VDS-dsRNAs and AV-Bc-VDS-dsRNAs (4:1) on leaves was assessed in two replicate experiments by extracting total RNA from leaves followed by a northern blot assay with probes specific to the Bc-VDS-dsRNA. 4-week-old Arabidopsis plants were treated at day 0 with either a 20 μl drop of Bc-VPS51+DCTN1+SAC1-dsRNAs (20 ng μl⁻¹) or AV-Bc-VDS-dsRNAs (400:100 ng μl⁻¹) and maintained under greenhouse conditions. Single leaf samples were collected at 1, 3, 7, and 10 dpt. Total RNA was extracted using TRIzol and subjected to northern blot analysis.

The potential environmental degradation of dsRNA was investigated by exposure of naked‐Bc VPS51 + DCTN + SAC1‐dsRNA (200 ng) and AV‐Bc‐VDS‐dsRNA (200 ng/2.5 μg) to Micrococcal Nuclease enzyme (MNase) (Thermo Fisher) treatment in four replicate experiments. Samples were treated with 0.2 U/μL MNase for 10 min at 37 °C, and dsRNAs were released using 1% Triton X‐100. All samples were visualized on a 2% agarose gel. The persistence of sprayed naked‐Bc VDS‐dsRNAs and AV‐Bc‐VDS‐dsRNAs (4:1) on leaves was assessed in two replicate experiments by extracting total RNA from leaves followed by a northern blot assay with probes specific to the Bc‐VDS‐dsRNA. 4‐week‐old Arabidopsis plants were treated at day 0 with either a 20 μL drop of Bc‐VPS51 + DCTN1 + SAC1‐dsRNAs (20 ng/μL) or AV‐Bc‐VDS‐dsRNAs (400:100 ng/μL) and maintained under greenhouse conditions. Single leaf samples were collected at 1, 3, 7 and 10 dpt. Total RNA was extracted using TRIzol and subjected to northern blot analysis.