Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing Qing Luo a,b , Quan Kang a,b , Wen-Xin Song b , Hue H. Luu b , Xiaoji Luo a,b , Naili An c , Jinyong Luo a,b , Zhong-Liang Deng a,b , Wei Jiang b , Hong Yin b , Jin Chen a,b , Katie A. Sharff b , Ni Tang a,b , Erwin Bennett b , Rex C. Haydon b , Tong-Chuan He a,b, ⁎ a The Children’s Hospital, and the Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing University of Medical Sciences, Chongqing 400016, China b Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC3079, Chicago, IL 60637, USA c The Scripps Research Institute, La Jolla, CA 92037, USA Received 23 November 2006; received in revised form 13 February 2007; accepted 15 February 2007 Received by A. Rynditch Available online 23 March 2007 Abstract RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human β-catenin, as well as with the inhibition of β-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites. © 2007 Elsevier B.V. All rights reserved. Keywords: Beta-catenin signaling; Expression knockdown; RNA interference; Short interfering RNA; Wnt signaling 1. Introduction Double-stranded RNA (dsRNA) can induce gene-silencing processes in eukaryotes through the degradation of homologous mRNAs, a process known as RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants (Sharp, 1999; Bosher and Labouesse, 2000; Zamore, 2001; Hannon, 2002; Paddison and Hannon, 2002; Tijsterman et al., 2002; Dykxhoorn et al., 2003; Mello and Conte, 2004; Montgomery, 2004; Tian et al., 2004). Small synthetic 19–27 nucleotide dsRNAs can act as short interfering RNAs (siRNAs) to mediate gene-specific silencing in mammalian cells (Sharp, 1999; Bosher and Labouesse, 2000; Zamore, 2001; Hannon, 2002; Paddison and Hannon, 2002; Tijsterman et al., 2002; Dykxhoorn et al., 2003; Scherer and Rossi, 2003; Mello and Conte, 2004). Practical applications of RNAi have been facilitated by using expression vector-based strategies to introduce siRNAs, commonly referred to as short hairpin RNAs (shRNAs), into mammalian cells using Pol III-dependent promoters (Paul et al., 2002; Scherer and Rossi, 2003; Medema, 2004; Aravin and Tuschl, 2005; Arziman et al., 2005; Bentwich, 2005; Dillon et al., 2005; Huppi et al., 2005; Sandy et al., 2005; Echeverri and Perrimon, 2006). To date, two RNA polymerase Gene 395 (2007) 160 – 169 www.elsevier.com/locate/gene Abbreviations: BC, β-catenin; GFP, green fluorescent protein; H1, RNA polymerase III (Pol III)-dependent promoter of the human H1 transcript of the nuclear RNase P complex; RFP, (monomeric) red fluorescent protein; RNAi, RNA interference; siRNA, short interfering RNA; U6, RNA polymerase III (Pol III)-dependent promoter of the human U1 small nuclear RNA (snRNA) gene. ⁎ Corresponding author. Tel.: +1 773 702 7169; fax: +1 773 834 4598. E-mail address: tche@surgery.bsd.uchicago.edu (T.-C. He). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.02.030