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Fig. 3 | Molecular Neurodegeneration

Fig. 3

From: Functional microRNA targetome undergoes degeneration-induced shift in the retina

Fig. 3

AGO2 HITS-CLIP workflow in mouse retina. (A) Mice were subjected to 5 days of photo-oxidative damage, dim-reared mice were used as control. Retinas were excised from eyeball and diced into a fine suspension, which was UV cross-linked. Tissue was lysed, digested with RNase and AGO2-containing complexes immunoprecipitated as described previously (Moore et al. 2014). (B) RNA of purified complexes was radiolabeled by 3′ linker ligation as previously described (Moore et al. 2014). Complexes were separated by SDS-PAGE, transferred to membrane and exposed to film. Autoradiogram showing 110 kDa and 130 kDa complexes representing the AGO2:miRNA and AGO2:miRNA:mRNA complexes, respectively. No AGO2 antibody (green box) and RNase over-digested (blue box) controls were included. (C) To maximise yield, RNA was extracted from complexes by on-bead proteinase K treatment. Total RNA was purified by phenol/chloroform extraction and the supernatant further purified using the mirVana Kit to separate (D) mRNA. (E) Positional read coverage of key genes within the rhodopsin pathway shows considerable homogeneity between intra-group samples. (F) Small RNA was isolated for miRNA analysis

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