Bacteria grown under the required growth conditions were pelleted and RNA was isolated using the SV total RNA purification kit (Promega) as described [34 (link)]. Total RNA (20 μg) was separated on MOPS agarose gels (1.2%), transferred by vacuum blotting for 1.5 h onto positively charged membranes (Whatman) in 10 x SSC buffer (1.5 M NaCl, 0.15 M sodium citrate, pH7) using a semi-dry blotting system and UV cross-linked. Prehybridization, hybridization to DIG-labelled probes and membrane washing were conducted using the DIG Luminescent Detection Kit (Roche, Germany) according to the manufacturer's instructions. The DIG-labelled PCR fragments used as probes were produced by PCR using the DIG-PCR nucleotide mix (Roche, Germany) as described [34 (link)] with the following primer pairs: for the lcrF transcript—I214/I303, for the csrB and csrC transcripts—555/556 and 583/I82, for the rne transcript—IV529/IV530 and the pnp transcript—IV527/IV528 (see S2 Table ).
To determine stability of the lcrF transcript, RNA stability assays were performed. In order to stop the de novo mRNA synthesis 0.5 mg/ml rifampicin (Serva) was added. 0, 1, 2, 3, 5 and 7.5 min after rifampicin treatment, 10% v/v phenol was added and the samples were snap frozen in liquid nitrogen. RNA isolation and northern blot analysis were performed as described above.
To determine stability of the lcrF transcript, RNA stability assays were performed. In order to stop the de novo mRNA synthesis 0.5 mg/ml rifampicin (Serva) was added. 0, 1, 2, 3, 5 and 7.5 min after rifampicin treatment, 10% v/v phenol was added and the samples were snap frozen in liquid nitrogen. RNA isolation and northern blot analysis were performed as described above.
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