To obtain total RNA from L. plantarum IMDO 130201, 5 mL of fermentation medium of a mid-exponential growth phase culture, was collected in 10 mL RNAprotect (Qiagen, Hilden, Germany), mixed, and kept at room temperature for minimum 5 min. Subsequently, the sample was centrifuged at 5,000 Í g for 15 min and the RNA was isolated from the resulting cell pellet by applying an enzymatic lysis using mutanolysin and lysozyme, after which the RNA was extracted from the resulting mixture by using an RNeasy minikit (Qiagen) following standard instructions and including mechanical disruption of the cells using glass beads, as described previously (Weckx et al., 2009). As the RNA profiles, obtained after capillary electrophoresis using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) showed an unexpected peak at the lower size range, possibly due to the presence of small RNA molecules (e.g., tRNA), an additional RNA clean-up step was performed. To remove the small RNA molecules, the Microcon YM-50 (Millipore, Bedford, MA) was used according to the Manufacturer’s standard instructions. Sampling was performed in duplicate, resulting in two technical repeat samples for each pH value.
Label
Cy5
Label protocol
RNA was linearly amplified using Genisphere SensAmp kit (Genisphere, Hatfield, Pennsylvania, USA) using 200 ng of total RNA. The protocol was followed according to manufacturer's instructions. The amplified RNA (aRNA) was purified using the RNeasy Mini Kit (Qiagen), and the purified aRNA was Cy5 labeled by a reverse transcription reaction as earlier described by Puskas LG, Zvara A, Hackler L, Van Hummelen P: RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 2002, 32(6):1330-1340.
To obtain total RNA from L. plantarum IMDO 130201, 5 mL of fermentation medium of a mid-exponential growth phase culture, was collected in 10 mL RNAprotect (Qiagen, Hilden, Germany), mixed, and kept at room temperature for minimum 5 min. Subsequently, the sample was centrifuged at 5,000 Í g for 15 min and the RNA was isolated from the resulting cell pellet by applying an enzymatic lysis using mutanolysin and lysozyme, after which the RNA was extracted from the resulting mixture by using an RNeasy minikit (Qiagen) following standard instructions and including mechanical disruption of the cells using glass beads, as described previously (Weckx et al., 2009). As the RNA profiles, obtained after capillary electrophoresis using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) showed an unexpected peak at the lower size range, possibly due to the presence of small RNA molecules (e.g., tRNA), an additional RNA clean-up step was performed. To remove the small RNA molecules, the Microcon YM-50 (Millipore, Bedford, MA) was used according to the Manufacturer’s standard instructions. Sampling was performed in duplicate, resulting in two technical repeat samples for each pH value.
Label
Cy3
Label protocol
RNA was linearly amplified using Genisphere SensAmp kit (Genisphere, Hatfield, Pennsylvania, USA) using 200 ng of total RNA. The protocol was followed according to manufacturer's instructions. The amplified RNA (aRNA) was purified using the RNeasy Mini Kit (Qiagen), and the purified aRNA was Cy3 labeled by a reverse transcription reaction as earlier described by Puskas LG, Zvara A, Hackler L, Van Hummelen P: RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 2002, 32(6):1330-1340.
Hybridization protocol
Samples were hybridized for 16 h using a HS 4800 Pro automated hybridization station (Tecan Systems Inc., San Jose, CA), as described previously (Weckx et al., 2009), except that with the only difference that 130 µL hybridization mixture was used instead of 210 µL.
Scan protocol
Slides were scanned using the Agilent scanner (Agilent) and images were analysed using ArrayVision v7 (GE Healthcare).
Description
The bacterial strain used throughout this study was L. plantarum IMDO 130201, an isolate obtained from a 10-day laboratory wheat sourdough fermentation performed through back-slopping (Van der Meulen et al., 2007). The strain was stored at –80°C in wheat sourdough simulation medium (W-SSM; Vrancken et al., 2008), supplemented with 25 % (vol/vol) glycerol as cryoprotectant. W-SSM was also used as the medium to perform simulated wheat sourdough fermentations.
Data processing
We apply a background correction, i.e. we subtract the local background for the local foreground intensities. We compute the base 2 log-ratios (ratio of Cy5/Cy3). Per slide, a Loess correction per print tip on the average log-ratios was applied, to remove non-linear dye effects and print-tip effects per array. We average the log-ratios over the replicates (# = 3) on the array.
Lactobacillus plantarum IMDO 130201, a wheat sourdough isolate, adapts to growth in wheat sourdough simulation medium at different pH values through differential gene expression
