RNA isolation and labelling for DNA microarray

Total bacterial RNA was isolated from bacteria grown under different growth conditions after killing by the addition of 0.2 volumes of 95% ethanol, 5% phenol, pH 4.3. Pellets were resuspended in 10 mM Tris, 1 mM EDTA containing 2 mg ml-1 lysozyme and incubated at 37°C for 30 min. Cell lysis solution (Qiagen, Hilden, Germany) was added and the mixture was incubated at 65°C for 5 min and at room temperature for 10 min. After the addition of precipitation solution (Qiagen, Hilden, Germany) and incubation on ice for 5 min cell debris, proteins and DNA were pelleted. The RNA containing supernatant was mixed with ethanol and loaded on a spin column (Promega). Further RNA purification and DNase digestion was done as described by the manufacturer.

A total of 50 μg of RNA of six separate experiments was reverse transcribed to cDNA and labelled with Cy3- or Cy5-conjugated dCTP (GE Healthcare) using reverse transcriptase (SupersciptII, Invitrogen) and random hexamers as primers. RNA was removed by hot-alkali treatment. Labelled cDNA was purified using a Qiaquick PCR purification kit and quantified by Nano-Drop analysis (ND-1000 Spectrophotometer, Peqlab).