RNA was isolated from strains HV30 × pTA232-tele-0582anti#1, -#2, and -#3 and wild-type strain HV30 × pTA232 as described before (64 (link)). Ten micrograms of RNA was separated on a denaturing 8% polyacrylamide gel or a denaturing 1% agarose gel and transferred to a nylon membrane (Hybond-N+ membrane or Pall membrane). For hybridization experiments, radioactively labeled PCR probes against the desired targets were generated using the DECAprime II DNA labeling kit and [α-32P]dCTP (Life Technologies, Thermo Fisher Scientific). Membranes were subsequently incubated with the labeled PCR fragments.
To quantify the repression efficiency, Northern blotting membranes were exposed to imaging plates and analyzed with the Amersham Typhoon biomolecular imager and ImageQuantTL software. Signals were compared with the signals for the 16S rRNA used as a loading control. The amount of RNA signals detected for the wild-type controls was set to 100%. Northern blot analyses were done in triplicates.
To quantify the repression efficiency, Northern blotting membranes were exposed to imaging plates and analyzed with the Amersham Typhoon biomolecular imager and ImageQuantTL software. Signals were compared with the signals for the 16S rRNA used as a loading control. The amount of RNA signals detected for the wild-type controls was set to 100%. Northern blot analyses were done in triplicates.
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