Ambion silencer select sirna

Ambion Silencer Select siRNA (Invitrogen) was used for corneal knockdown experiments and transfection mediated using lipofectamine 2000 (Invitrogen) as described previously.^{8 (link)} Briefly, partial epithelial debridement of corneas of anesthetized WT mice was conducted to prepare the eye for transfection. Next, 3.33 µmol tetherin-specific or nonspecific scramble control siRNA in supplement-free DMEM media was applied to each cornea in a 10 µl eye drop containing 5 µl Lipofecamine 2000 (Invitrogen). siRNA sequences were designed and validated by the manufacturer. Mice were infected with HSV-1 16 hours post-transfection. On days 2, 3, and 5 pi, tissue was harvested for plaque assay and validation of knockdown efficiency. Tetherin transcript expression was quantified using PrimePCR technology (Biorad) as described for the gene array. Tetherin protein expression was imaged in immunolabeled cornea flat mounts by confocal microscopy to evaluate knockdown efficiency.

For corneal FcRn knockdown experiments, Ambion Silencer Select siRNA
(Invitrogen) was used as previously described with Lipofectamine RNAiMax
(Invitrogen) tranfection reagent (33 (link)).
Briefly, the apical corneal epithelium was partially debrided to facilitate
efficient transfection and a drop containing 5 μL lipofectamine, and
3.33 nmol FcRn-specific or nonspecific scramble control siRNAs applied to each
cornea in supplement-free Dulbecco’s modified Eagle’s media.
siRNA sequences were designed and validated by the manufacturer, although FcRn
was targeted with two non-overlapping siRNAs to enhance effectiveness (Table S1). Knockdown
efficiency was confirmed by Western blot.