Mechanistic exploration of autophagy and aging by RNA interference

Cellular senescence is a cellular process with organismal impact that is mechanistically counterbalanced to a certain extent by frequent episodes of autophagy. Here we describe a detailed, automation-compatible method for the use of RNA-interference (RNAi; also called post-transcriptional gene silencing (PTGS))-mediated silencing of autophagy related protein-coding gene expression. RNAi is a conserved biological response to double-stranded RNA that mediates resistance to endogenous parasites and exogenous pathogenic nucleic acids. RNAi mediated by short interfering RNA (siRNA) is widely used for gene function analysis. The accurate use of RNAi for the inference of gene function necessitates that both specificity and efficacy of the siRNA-mediated knockdown are monitored. In this manuscript, we exemplify these crucial steps employing siRNAs targeting the autophagy and lysosomal biogenesis associated transcription factor TFE3 and validate their specificity on protein and mRNA level.