Quantitative RT-PCR for agrB Gene Expression

The wild-type parent ATCC 3624 and derivatives (the agrB-null mutant or complementing strain) were grown in TY medium for 5 h at 37°C. Cultures were then pelleted and RNA was extracted using saturated phenol and purified by TRIzol and chloroform (Life Technology and Sigma), as previously described (36 (link)). After the absence of DNA was confirmed (36 (link)) by subjecting samples to PCR without reverse transcriptase, RNA was quantified by measuring the sample absorbance at 260 nm. An aliquot of 1 μl of purified RNA (100 ng) was then used in a one-step RT-PCR containing 10 μl of 2× Taq master mix (New England Biolabs), 4 U of avian myeloblastosis virus reverse transcriptase (Promega), and agrB gene primers (described earlier), with ddH₂O added to reach a 20-μl total volume. Similarly, 16S RNA RT-PCR was performed as a loading control (36 (link)). Reaction mixtures were incubated for 45 min at 45°C to allow cDNA synthesis, and then regular PCR cycling was performed using the following conditions: (i) 95°C for 2 min; (ii) 30 cycles of 95°C for 15s, 50°C for 30 s, and 68°C for 30 s; and (iii) a final extension of 68°C for 5 min.