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. Author manuscript; available in PMC: 2013 Dec 21.
Published in final edited form as: Invest Ophthalmol Vis Sci. 2013 Jan 17;54(1):10.1167/iovs.12-10528. doi: 10.1167/iovs.12-10528

Figure 6. 5′RACE verifies siRNA-mediated cleavage at the predicted site on K12 mRNA.

Figure 6

K12 specific mRNA cleavage products of AD293 cells exogenously expressing K12-Arg135Thr and treated with 3nM siRNA K12-Arg135Thr-5 were assessed by a modified 5′ Rapid Amplification of cDNA Ends (5′RACE). The generacer oligo adapter was ligated to the free 5′ end of cleaved mRNA products. K12 specific cleaved mRNAs were selected by PCR amplification with a K12 Gene Specific Primer (GSP). The major K12 specific PCR product was purified, ligated into pCR4.0 topo plasmid and sequenced. Sequencing shows that the major product had the generacer adapter ligated to the 399th nucleotide of the K12 CDS, this corresponding to the predicted cleavage site between nucleotides 10 and 11 of the hybridized guide strand of siRNA K12-Arg135Thr-5.