Figure 2.

shRNAmir design and cloning. (a) Example of shRNA guide strand predictions (from DSIR or similar tool) screened against a series of Sensor exclusion criteria (gray box). The example shows two potential 21-mer predictions targeting the Renilla luciferase cDNA. Each shRNAis given a numerical designation (e.g., Ren.713) that reflects the first nucleotide position of the target sequence in the mRNA transcript. Sequences that pass all criteria are selected for cloning and testing in vitro. (b) Schematic overview of the process to transform 21-mer guide strand predictions into miR30-based cloning templates for PCR (upper box) or linker (lower box) cloning. First, the 21-mer guide strand (gray) is reverse complemented to generate the 21-mer sense strand (or 21-mer target site; green). To generate the appropriate shRNAmir template, the nucleotide immediately 5′ to the 21-mer sense strand (orange) is changed according to the nucleotide 5′ to the 21-mer target site in the mRNA transcript (gray); if the 5′ nucleotide in the mRNAis an A or U, the first base of the 22-mer sense strand becomes a C, and if the 5′ nucleotide in the mRNAis a C or G, the first base of the 22-mer sense strand becomes an A. The final 22-mer sense strand is then inserted into a 97-mer (PCR) or 110-mer (linker) cloning template (Table 1). The 22-bp guide strand is the exact reverse complement of the 22-bp target site. XhoI/EcoRI cloning fragments are then generated by PCR amplification using specific primers (Table 1) or by annealing two complementary oligonucleotides (linker cloning). Pos., position.