Figure 3.—
Northern blot analyses to compare the relative amount of aadA hairpin RNA and siRNA for RNAi-37 subclones with various levels of spectinomycin resistance. (A) Evaluation of spectinomycin resistance for six RNAi-37 clones. Three strongly silenced subclones (nos. 1–3) and weakly silenced subclones (nos. 4–6) were spotted on various kinds of plates after serial dilutions of 10−1, 10−2, and 10−3. (B) Northern blot analyses to detect aadA hairpin RNA and siRNA. A sense-aadA RNA probe was used to detect the hairpin RNA and siRNA. Total RNA was loaded on a 1% agarose/formaldehyde gel and a small-sized RNA fraction was resolved on a 15% polyacrylamide gel containing 8 m urea. Ethidium bromide staining and detection of the RbcS2 mRNA were performed to confirm that the amount of RNA loaded was almost equal. Hybridization using the RbcS2 RNA probe was carried out last, since complete stripping of the probe was difficult.