Transcriptomic changes associated with oxidative stress in the newt retinal pigment epithelial cells

Molecular mechanisms of the Retinal Pigment Epithelial cells response to different stress conditions in the newt remain unexplored. It is assumed that the features of endogenous defense systems in Urodela make RPE resistant to oxidative stress and associated pathologies. The goal of our project is aimed to identify the key contributors of endogenous defense cell systems in RPE in the adult newt P. waltl, after simulated the conditions of oxidative stress. The main task is related to the study of the RPE transcriptome changes in response to H2O2, compared the norm. We have sequenced the RPE cells transcriptome, using the RNA-Seq technology. Total RNA from RPE was extracted using the "Extract RNA" which utilizes guanidine thiocyanate (Cat. No. BCO32, Evrogen, RF). DNA was removed with TURBO DNA-free (Thermo Fisher Scientific, Waltham, MA, USA). For sequencing, 400 ng of RNA was taken from each sample. RNA quality and integrity were assessed at each step using an Agilent 2100 Bioanalyzer (RIN value greater than seven) (Agilent Genomics, Santa Clara, CA).De novo transcriptome assembly (obtaining sequences of sequenced transcripts) preceded the assessment of the molecular profile of the RPE, and was carried out using the Trinity package program (https://github.com/trinityrnaseq/trinityrnaseq/wiki).Identification of the signaling and metabolic pathways associated to the main molecular components of the endogenous defense system in the newt RPE will help to look into the mechanisms of retinal diseases (proliferative vitreoretinopathy, et al.) and to find an acceptable approaches to prevent the retinal neurodegeneration in human.