Journal: Cell Death & Disease
Article Title: Ocular neuroprotection by siRNA targeting caspase-2
Figure Lengend Snippet: Localization, stability and RNAi activity of intravitreally injected siCASP2. ( a ) Detection of Cy3 fluorescence in isolated RGC 1 h (red line) or 18 h (green line) following single intravitreal injection of 40 μ g Cy3-siRNA per rat eye. FACS analysis displaying results obtained from representative one out of two retinal tissue pools analysed per time point are shown. ( b ) Representative micro-autoradiographs of paraffin sections (counterstained with haematoxylin and eosin) from untreated ((i), (ii)) and siCASP2-injected rat eyes ((iii)–(iv)), which were hybridized with either siCASP2-specific ((i)–(iv)) or with a nonspecific ((v), (vi)) control 33 P-labelled probe ((i), (iii), (v)) – bright field images, ((ii), (iv), (vi)) – corresponding dark field images. In dark field images, hybridization signals are white dots over all retinal layers in sections of siCASP2-injected eyes hybridized to the specific probe ((iv)) and were especially prominent in the GCL (arrow). No signal in the GCL is detected in control sections ((ii), (vi)) (exposure 24 h, original magnification × 20). The eyes for in situ hybridization analysis were enucleated at 2 h after intravitreal injection of 20 μ g siRNA. At least 10 eyes from each group were analysed from 2 to 3 different experiments. ( c ) Quantification of siCASP2±S.D. in retina at different times after intravitreal injection of 20 μ g per eye siCASP2 ( n =6). Control retinas were obtained from intact non-injected eyes. ( d ) Detection of siCASP2-mediated RNAi using RLM-RACE: (i) EtBr-stained agarose gel with electrophoresed RLM-RACE products from siCASP2-transfected PC12 cells (size control) and from retinas collected at 4 h after intravitreal injection of PBS (1); 20 μ g siCNL (2); or 20 μ g siCASP2 (3) (yellow boxes indicate gel regions excised for cloning and subsequent colony sequencing); (ii) autoradiograph of the Southern blot of the gel shown in (i) after hybridization with radiolabelled RLM-RACE junction-specific probe; and (iii) example of the results of colony sequencing showing the RLM-RACE junction corresponding to the expected siCASP2 produced cleavage site. RLM-RACE was performed on RNA extracted from a pool of two retinas from eyes injected with PBS, pool of five retinas from the eyes injected with siCNL or a pool of four retinas from the eyes injected with siCASP2
Article Snippet: Whole retinas were washed in a large volume of ice-cold PBS and RNA was extracted using EZ-RNA RNA extraction kit (Biological Industries, Kibbutz Beit-Haemek, Israel).
Techniques: Activity Assay, Injection, Fluorescence, Isolation, FACS, Hybridization, In Situ Hybridization, Staining, Agarose Gel Electrophoresis, Transfection, Clone Assay, Sequencing, Autoradiography, Southern Blot, Produced