miR-183 cluster gene includes three miR-183, miR-96 and miR-182 genes that in the adult retina are highly expressed in the inner nuclear layer and specifically in photoreceptors, and plays critical roles in development, maturation and normal function of the adult retina. Additionally down- or dis-regulation of miR-183/96/182 genes have been shown in retinal degenerative diseases such as retinitis pigmentosa. So, exogenously over-expression of miR-183 cluster genes seems to potentially induce trans-differentiation of potent cells like RPE into retinal neurons. With respect to this aim, following constructing of AAV-based vectors containing individual miR-183 cluster genes, the expression level of miR-183/96/182 was evaluated.

HEK-293T cells were transfected with the constructs containing each miR-183/96/182 using calcium phosphate precipitation method. After 48 hours, total RNA was prepared using Tripure isolation reagent and treated with DNase for eliminating genomic DNA. cDNAs were synthesized by stem-loop primers specifically designed for each miR-183/96/182 and then applied to perform stem-loop RT-qPCR method for measuring individual miRNAs. To ensure that the amplified fragments were actually miR-183/96/183, sequencing was done.

Using stem-loop RT-qPCR method, the expression of miR-183 cluster genes was detected and sequencing data verified the true identity of the observed PCR products as members of miR-183 cluster.

Our results promised that constructed miR-183/96/182 cluster would be efficiently working for probing their effects individually or in a tandem cluster in adult retinal cells fate in vitro or in vivo.