First-Strand cDNA Synthesis Kit

Protocol tips

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	Heat the RNA solution to 65ºC for 10 minutes, then chill on ice. Add first-strand cDNA reaction mix and incubate at 37°C for 1 hour

Protocol tips
Heat the RNA solution to 65ºC for 10 minutes, then chill on ice. Add first-strand cDNA reaction mix and incubate at 37°C for 1 hour

Publication protocol

Bacteria were harvested during the stationary phase and RNA was extracted using the RNeasy Midi Kit (Qiagen). The purity and concentration of the RNA were determined by gel electrophoresis and spectrophotometry (NanoDrop, Thermo Scientific). First-strand cDNA was reverse transcribed from 1 µg of total RNA with the First-strand cDNA Synthesis Kit (Amersham Biosciences, GE Healthcare). Quantitative real-time PCR (qRT-PCR) was performed in a Bio-Rad iCycler with Bio-Rad iQ SYBR Green Supermix. For all primer sets (Supplementary Table S4), the following cycling parameters were used: 94°C for 3 min followed by 40 cycles of 94°C for 60 s, 55°C for 45 s and 72°C for 60 s, followed by 72°C for 7 min. oprI (house-keeping gene control, outer membrane lipoprotein precursor) was used to normalize gene expression (29). Amplification products were electrophoresed on 0.8% agarose gels. For statistical analysis of relative gene expression, the 2−ΔΔCT method was used (30).

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