TGA is independent of the cleavage activity of Ago2 and the levels of steady-state promoter-associated RNAs do not correlate to TGA. (A) Strand-specific qRT-PCR to detect promoter-associated antisense RNA in clones C5, D1, and E3. Standard curves were generated from genomic DNA standards of the same gene. Data are normalized as cDNA copies per cell, with the assumption that 1 μg cDNA corresponds to 105 cells. (B) Strand-specific qRT-PCR for the antisense RNA from clone C5 4 d after treatment with irrelevant shRNA (ctrl-shRNA) or the TGA-inducing shRNAs (shRNA-(0)AS and shRNA-(-4)). Statistical analysis in this study was performed using a two-tailed t-test. (C) Strand-specific qRT-PCR for the sense eGFP mRNA from clone C5 4 d after treatment of irrelevant shRNA (ctrl-shRNA) or three mutated variants of shRNA-(0). The strand of shRNA-(0)AS is mutated at nucleotides 1–5 (shRNA-(0)AS-M5′), nucleotides 9–11 (shRNA-(0)AS-M(9-11)), or nucleotides 15–19 (shRNA-(0)AS-M3′). Sequences for the shRNAs are provided in Supplemental Table S2.
