Silencer sirna labelling kit

SsGST-mu1 dsRNA was labelled using the Silencer™ siRNA labelling kit (Thermo Fischer Scientific). Ten μl labelling dye (Cy3®), 12.5 μg SsGST-mu1 dsRNA in 15 μl normal saline, 5 μl 10× labelling buffer and 20 μl nuclease free water (Invitrogen, Carlsbad, USA) were combined and incubated at 37 °C for 3 h. To precipitate the dsRNA 5 μl 5 M NaCl and 125 μl cold 100% ethanol were mixed with the reaction and incubated 30 min at -20 °C. The dsRNA was pelleted by centrifugation at 18,400×g for 20 min at 4 °C, washed with 70% ethanol and centrifuged for 5 min at the room temperature, air - dried and resuspended in 10 μl of nuclease free normal saline (Sigma-Aldrich). Successful labelling was indicated by pink staining of the pellet and confirmed by agarose gel electrophoresis.

Synthetic miRNA was labelled with Cy3 using the Silencer siRNA labelling kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Five-day-old Col-0 seedlings grown in vertical plates (MS half strength, 0.5% sucrose and 0.7% plant agar) were treated by applying 9.6 µl of the labelled miRNA (20 µM) along the roots. After 2 h incubation, confocal microscopy was performed. Samples were washed two or three times with distilled water to prevent labelled miRNA from attaching to the outer root surface. To verify that the fluorescence was emitted by the miRNA and not the free dye, a blank labelling reaction was performed (using water instead of miRNA), and plants were treated with this blank reaction. Treated roots were imaged using a Zeiss Airyscan 800 laser scanning confocal microscope. Cy3 miRNA was excited using a 561 nm laser and fluorescence was detected at 564–620 nm. pSUC2:YFP and pS18:YFP fluorescence was detected using a 488 nm laser and 492–550 nm emission wavelength. Sequential scanning was used to eliminate bleed-through.

Ec-LacZ dsRNA was labelled with the dye Cy3^® [10 (link)] using the Silencer™ siRNA labelling kit (Thermo Fischer Scientific, Waltham, MA, USA). Twenty-five each of pre-treated or untreated eggs were incubated in 2.5 µg/µL of Cy3^® labelled Ec-LacZ dsRNA in 0.9% nuclease free NaCl at 4 °C for 24 or 48 h or at room temperature (22 °C) for 24 h. Incubated eggs were washed by dipping them 3 times into PBS-0.05% Tween (PBST) and once into PBS to remove excess dsRNA before examination under a Zeiss 780-NLO confocal microscope with a 20× Plan-Apochromat (NA 0.8) lens. Z stack images (4–8 slices) were captured. Cy3^® was excited using a 561 nm laser attenuated 97% and emitted light (565–631 nm) was captured using a GaAsP array detector. Images are presented as a single representative slice through each egg processed using Zen software (Carl Zeiss AG, Oberkochen, Germany). Eggs with developed larvae were excluded, to eliminate false positive signals due to autofluorescence of the S. scabiei cuticle. The dsRNA uptake efficiency was determined as the percentage of fluorescent positive eggs of the total number of eggs incubated.