Figure 2

Short and long shRNAs are both potent suppressors. (A) The suppressive activities of a set of 17 related hairpins with 15, 17 - 29, 33, 37, and 41 bp stem lengths were tested. n.b. for hairpins longer than 29 bp, position 30 (1) was altered to a 'G' to prevent a run of 'T's which would have resulted in premature termination of transcription, however, this was not expected to impact on suppressive activity. Three sets of data are shown; specific suppressive activity in the top (as a decrease in fluorescence of the GFP-target fusion), non-specific shRNA effects in the second row graph (represented by the normalization factor), and standard expression anlysis of the processed siRNA products (15% PAGE) on the third row. (B) Additional high resolution northern analysis (20% PAGE) for the same hairpins. (C) 293a cells were transfected with increasing amounts of hairpin vector from 0.1 ng to 400 ng, for the Tat56-19, 21, 23, 25, 27 and 29 bp hairpins. Each reaction was supplemented with the appropriate amount of empty expression vector to keep the total expression vector DNA delivered at 400 ng. All activity values are averages from at least 3 independently repeated experiments with 95% Confidence Intervals (CI) shown.