Design of siRNAs and shRNAs. The R4 siRNA was designed to target IGF1R mRNA at
TG101348 639–657, which was identified in our empirical array-based screen as a highly accessible region of the IGF1R transcript [7]. The sequences of the R4 IGF1R siRNA and the corresponding non-silencing control duplex (ScrR4) have been previously described [8]. The E1 duplex targeted EGFR mRNA at nucleotides 343–361 and was identified during an array-based screen of in vitro transcribed siRNA (M. Sohail, unpublished). The E1 duplex has the sequence: sense strand 5′-GGCACGAGUAACAAGCUCATT-3′ and antisense strand 5′-UGAGCUUGUUACUCGUGCCTT-3′. The sequence of the scrambled control duplex (ScrE1) was: sense strand 5′-AGAUAACCGAACAAGCGUGTT-3′ and antisense strand 5′-CACGCUUGUUCGGUUAUCUTT-3′. Each siRNA strand incorporated 19 bases of RNA with two 3′-deoxythymidines. Oligonucleotides were synthesised, HPLC/HPP purified and annealed at Qiagen (Germany). All sequences were submitted to BLAST and Smith–Waterman searches to ensure that R4 was specific for the IGF1R and E1 for the EGFR, and that control sequences ScrR4 and ScrE1 were not homologous to any known genes. To induce stable long-term RNAi-mediated knockdown of the IGF1R gene, pSUPER
plasmids were constructed to express short hairpin RNAs (shRNAs) as described [9]. Phosphorylated and annealed 64-mer double-strand DNA oligonucleotides incorporating BglII and HindIII overhangs were ligated into linearised pSUPER vector containing compatible overhangs. The sequence of the IGF1R shRNA was designed to target the same region of the transcript as the R4 siRNA duplex, and the scrambled control shRNA sequence was based on the non-silencing scrambled control siRNA ScrR4.
