Analyzing the effects of a gene-specific siRNA on IL13R-α2 mRNA and protein levels in glioblastoma multiforme cells INTRODUCTION  RNA interference (RNAi) - A post-transcriptional system found in most eukaryotes and animals that control the activity of genes (Figure 1), triggered by the presence of dsRNAs that are homologous to a portion of a specific gene (DeRuiter, S., et al. 2002).  Small-interfering RNA (siRNA) - a post-transcriptional enzyme called Dicer cleaves dsRNA into short RNA fragments of about 20 nt long, which are homologous to a portion of a specific gene and binds to its mRNA (Akinc, A., et al. 2004).  RNA-induced silencing complex (RISC) - siRNA bound to its homologous mRNA of a specific gene (Figure1).  Argonaute - A catalytic enzyme that recognizes the complex and cleaves it thus degrading the mRNA of the targeted gene (DeRuiter, S., et al. 2002).  Interleukin 13 receptor alpha 2 (IL13R- α 2)- a mutated transmembrane receptor protein expressed on several aggressive human cancers, including a common type of brain cancer cell called glioblastoma multiforme (Pope and Thompson 2008).  Create a vector containing siRNA of a specific IL13R- α 2 insert (Figure 3).  Transfect U87MG glioblastoma multiforme cells, and isolate total RNA and total protein.  Observe the down-regulation of IL13R- α 2 mRNA and protein via quantitative-PCR and western blotting, respectively (Figure 2). Using psiRNA Kit from Invivogen(Figure 3): -restriction digest of psiRNA-h7SKneo G1 plasmid vector -ligation of IL13R-α2 siRNA insert and scramble to the psiRNA Transfect U87MG glioblastoma cells with psiRNA using the LyoVec™ transfection kit (Figure 4). Isolate total RNA and protein from transfected U87MG cells with Trizol™ (Figure 4). BCA protein assay of total proteins. SDS PAGE of equal amounts of protein to observe IL13R-α2 protein activity in Western Blot (Figure 6). Reverse transcription of total RNA into cDNA using RetroScript™ Quantitative PCR of cDNA samples with gene- specific TaqMan reagents to observe IL13R-α2 and GAPDH mRNA levels in each sample (Figure7). with IL13R-α2 antibody (Figure 6) with GAPDH antibody (Figure 6) With IL13R-α2 primers (Figure 7) With GAPDH primers (Figure 7) PCR with IL13R-α2 and GAPDH primers, analyze presence of cDNA on Agarose gel (Figure 5). EXPERIMENTAL DESIGN Raisa J. Cheng and Dr. Jeffrey P. Thompson, Ph.D. Department of Biology, York College of Pennsylvania, York, PA RESULTS a.Normal U87MG- untreated b.Normal U87MG-treated with G418 c.Tumor of U87MG with siRNA insert- treated with G418 d.U87MG with siRNA insert- treated with G kD 50 kD U87mgCRP5CRP10SCRINSGAPIL13Rladder  There was more mRNA detected in the IL13R-α2 insert than normal U87MG and IL13R-α2 scramble.  There was a greater amount of CRP5 mRNA produced than the CRP10, although there should be more CRP10 produced.  The discrepancies in the CRP5 and CRP10 samples could be caused by the type of ribosomes are responsible for their translation process.  These data suggest that high levels of mRNA for a gene do not always yield high levels of translated protein.  A mixed pool was used that contained both down-regulated receptors and unaffected receptors. Further research would be to isolate IL13R-α2 mRNA and protein from subclones that have more specific populations of down-regulated receptor. CONCLUSIONS A Special thank you to Dr. Jeffrey P. Thompson for all his help and guidance. ACKNOWLEDGEMENTS 1. Akinc, A., et al Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 432: DeRuiter, Stacy L., et al RNA interference by expression of short- interfering RNAs and hairpin RNAs in mammalian cells. PNAS. 99: Pope, Chris R. and Thompson, Jeffrey P Investigating the Glycosylation of Interleukin 13 Receptor Alpha 2 Protein Expressed in Cancerous and Non-cancerous Cell Lines. Poster Presentation. York College of Pennsylvania Biology Department. Figure 1. Model of RNA interference mechanism. Figure 2. Figure 3. Vector containing IL13R- α 2 siRNA insert. Figure 4. Results of transfection (see description below). Figure 5. Results of Agarose Gel showing the presence of IL13R- α 2 and GAPDH cDNA in each sample. Figure 6. Western Blot results showing IL13R- α 2 protein levels in each sample. GAPDH protein levels also shown. Figure 7. Results of Q-PCR showing levels of IL13R- α 2 mRNA in each sample. WORKS CITED a. d.c. b. OBJECTIVES