Total RNA was isolated from C. difficile 630Δerm strain grown in TY medium either after 4 h, 6 h or 10 h of growth and from R20291 strain during late exponential growth phase (6 h) as previously described [22] (link). Starvation conditions correspond to a 1 h incubation of exponentially grown cells (6 h of growth) in PBS buffer at 37°C. Strains carrying pRPF185 derivatives were grown in TY medium in the presence of 250–500 ng/mL ATc and 7.5 µg/mL Tm for 7.5 h followed by RNA isolation. The cDNA synthesis by reverse transcription and quantitative real-time PCR analysis was performed as previously described [45] (link). In each sample, the relative expression for a gene was calculated relatively to the 16S gene [81] (link). The relative change in gene expression was recorded as the ratio of normalized target concentrations (ΔΔCt) [82] (link). Strand-specific RT-PCR analysis was performed as previously described [27] (link).
For Northern blot analysis, 5 µg of total RNA was separated on a denaturing 6% or 8% polyacrylamide gel containing 8 M urea, and transferred to Hybond-N+ membrane (Amersham) by electroblotting using the Trans-blot cell from Bio-Rad in 1× TBE buffer (89 mM Tris-base, 89 mM boric acid and 2 mM EDTA). Following UV-cross-linking of the samples to the membrane, prehybridization was carried out for 2 h at 42°C in 7 mL of prehybridization buffer ULTRAHyb (Ambion). Hybridization was performed overnight at 42°C in the same buffer in the presence of a [gamma-32P]-labeled DNA oligonucleotide probe. Alternatively, the probe was synthesized using PCR with 5′end-labeled primer complementary to RNA sequence. After hybridization, membranes were washed twice for 5 min in 50 mL 2× SSC (300 mM sodium chloride and 30 mM sodium citrate) 0.1% sodium dodecyl sulphate (SDS) buffer and twice for 15 min in 50 mL 0.1× SSC 0.1% SDS buffer. Radioactive signal was detected with a Typhoon system (Amersham). The size of the transcripts was estimated by comparison with RNA molecular weight standards (Invitrogen).
For Northern blot analysis, 5 µg of total RNA was separated on a denaturing 6% or 8% polyacrylamide gel containing 8 M urea, and transferred to Hybond-N+ membrane (Amersham) by electroblotting using the Trans-blot cell from Bio-Rad in 1× TBE buffer (89 mM Tris-base, 89 mM boric acid and 2 mM EDTA). Following UV-cross-linking of the samples to the membrane, prehybridization was carried out for 2 h at 42°C in 7 mL of prehybridization buffer ULTRAHyb (Ambion). Hybridization was performed overnight at 42°C in the same buffer in the presence of a [gamma-32P]-labeled DNA oligonucleotide probe. Alternatively, the probe was synthesized using PCR with 5′end-labeled primer complementary to RNA sequence. After hybridization, membranes were washed twice for 5 min in 50 mL 2× SSC (300 mM sodium chloride and 30 mM sodium citrate) 0.1% sodium dodecyl sulphate (SDS) buffer and twice for 15 min in 50 mL 0.1× SSC 0.1% SDS buffer. Radioactive signal was detected with a Typhoon system (Amersham). The size of the transcripts was estimated by comparison with RNA molecular weight standards (Invitrogen).
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