Figure 1

Electroporation and siRNA delivery in intact human islets. (A) Graph showing RT-qPCR results of RB knockdown following lipid or electroporation transfection. Intact human islets were transfected with non-targeting control siRNA (D-001810 ON-TARGETplus from Thermo Fisher/Dharmacon) or siRNA targeting RB (L-003296) using lipid (Dharmafect 1 from Thermo Fisher/Dharmacon) or electroporation (Neon Transfection System from Life Technologies), cultured for two days, and RT-qPCR was performed to assess knockdown. Error bars indicate SEM (n = 3 separate experiments). (B) Graph showing representative results of glucose stimulation of insulin secretion assay. Intact islets were electroporated with control, non-targeting siRNA at a range of voltages (x-axis) and a glucose stimulation of insulin secretion assay was performed 72 hours following electroporation. Media was collected after exposure to 3 mM glucose (light gray bars), then after switch to 20 mM glucose (gray bars), followed by return to 3 mM glucose (black bars), and total insulin (hatched bars). (C) Images showing morphological comparison between non-electroporated (left) and electroporated (right) islets. Images of islets stained with hematoxylin and eosin (top), immunostained for for c-peptide (red) and nuclei DAPI (blue)(middle), and after exposure to ethidium bromide to test viability 72 hours after electroporation (bottom).