Figure 2.

Standard denaturing northern blot using the classical ‘baking’ of the membrane at 80°C for RNA immobilization. Lanes 1–3: various amounts of the chemically synthesized pRNA 14-mer (with 5′-OH terminus; Figure 1A); lanes 4 and 5: 5 or 10 ng of the chemically synthesized pRNA 14-mer mixed with 5 µg of total RNA from B. subtilis before electrophoresis and membrane transfer; lanes 6–8: samples with varying amounts of total RNA from B. subtilis for the purpose of endogenous pRNA detection. Total cellular RNA was isolated 3 min after outgrowth from stationary phase using the hot phenol method; the 5′-digoxigenin-labeled LNA–DNA mixmer shown in Figure 1A was used as probe. (for details, see ‘Materials and Methods’ section).