M mlv reverse transcriptase

The midlog phase of bacterial cultures was treated with 1 μmol·ml⁻¹ oxacillin for 24 h and was subsequently used for RNA extraction [28 (link)]. Treatment with RNAse-free DNAse I (Sigma) was performed at 37°C for 2 h. The concentration and DNase-free quality of RNA samples were spectrophotometrically assessed and confirmed by the amplification of chromosomal bla_OXA-51 and 16S rRNA. Fifteen microliters of each RNA sample was reverse-transcribed in a final volume of 20 μl containing random hexamers, MMLV reverse transcriptase (Agilent) at 42^o C for 45 min.
Amplification of bla_OXA-51, bla_OXA-58, and 16S rRNA was performed in a final volume of 25 μl containing 5 μl cDNA, 3 mmol MgCl₂, 200 nmol dNTPs, 2 U h-Taq DNA polymerase (Solgent), 300 nmol of OXA-51/58-F/R primers, 150 nmol of OXA-51/58-P probes, 200 nmol of 16S-F/R primers, and 100 nmol of 16S-P probe (IDT). Primer and probe sequences are given in Table 1. Each real-time PCR was performed in triplicate on the Stratagene Mx3005P real-time PCR system (Agilent). The reaction mixture was incubated for 15 min at 95°C, followed by 40 cycles of 10 s at 95°C and 20 s at 60°C. Normalized expression of bla_OXA-51 and bla_OXA-58 genes was calculated relatively to the 16S rRNA reference gene according to the 2^-ΔΔCt method [29 (link)].