For RNA extractions, 10 mL of an overnight culture was collected in 40 mL RNAprotect (Qiagen, Hilden, Germany) that was 2:1 diluted with 1x PBS buffer (Invitrogen, Carlsbad, California, USA), mixed, and kept at room temperature for minimal 5 min. The sample was subsequently centrifuged at 5,000 g for 15 min. The pellet was resuspended in 200 µL TE buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 1.3 U/µL mutanolysin (Sigma-Aldrich, Milwaukee, Wisconsin, USA) and 50 µg/µL lysozym (Sigma-Aldrich), and incubated in a shaking water bath at 37°C for 1 h. According to the RNeasy Mini Kit protocol, 700 µL of RLT buffer containing 10µL/mL ²-mercaptoethanol was added to the lysate, and the solution was mixed and transferred to a tube containing approximate 50 mg of acid-washed beads (Sigma-Aldrich) that was vortexed for 5 min. The tube was centrifuged for 10 sec. in a microcentrifuge at full speed and 850 µL of supernatant was added to 590 µL 80% ethanol. 700 µL of this mixture was applied on the RNeasy Mini column. Manufacturer's standard instructions were followed from that point. RNA quality was checked by measuring the 260nm/280nm and 260nm/230nm ratios using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, Delaware, USA), and by electrophoresis using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, California, USA).
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.
For RNA extractions, 10 mL of an overnight culture was collected in 40 mL RNAprotect (Qiagen, Hilden, Germany) that was 2:1 diluted with 1x PBS buffer (Invitrogen, Carlsbad, California, USA), mixed, and kept at room temperature for minimal 5 min. The sample was subsequently centrifuged at 5,000 g for 15 min. The pellet was resuspended in 200 µL TE buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 1.3 U/µL mutanolysin (Sigma-Aldrich, Milwaukee, Wisconsin, USA) and 50 µg/µL lysozym (Sigma-Aldrich), and incubated in a shaking water bath at 37°C for 1 h. According to the RNeasy Mini Kit protocol, 700 µL of RLT buffer containing 10µL/mL ²-mercaptoethanol was added to the lysate, and the solution was mixed and transferred to a tube containing approximate 50 mg of acid-washed beads (Sigma-Aldrich) that was vortexed for 5 min. The tube was centrifuged for 10 sec. in a microcentrifuge at full speed and 850 µL of supernatant was added to 590 µL 80% ethanol. 700 µL of this mixture was applied on the RNeasy Mini column. Manufacturer's standard instructions were followed from that point. RNA quality was checked by measuring the 260nm/280nm and 260nm/230nm ratios using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, Delaware, USA), and by electrophoresis using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, California, USA).
Label
Cy3
Label protocol
RNA was linearly amplified using Genisphere's 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
Hybridization mixtures were prepared using 50 pmol labeled aRNA in 210 µL Hybridization Buffer (GE Healthcare) containing 50% formamide (Sigma-Aldrich). The hybridization mixtures were denaturized at 96C for 3 min, put on ice for at least 5 min, and were hold at 32C for 5 min and subsequently centrifuged at 12,000 rpm for 5 min. The samples were hybridized with an HS 4800 Pro Hybridization Station (Tecan Systems Inc, San Jose, USA) at 32C for 16 h. Post-hybridization washing (automated) was performed in 1x SSC / 0.2% SDS at 32C, 27C and 23C for 20sec, 20sec and 30sec, respectively. This was followed by 0.1x SSC / 0.2% SDS for 1 min at 23C and in 0.1x SSC at 23C for 30 sec. Slides were dried for 2 min at 30C using nitrogen gas.
Scan protocol
Slides were scanned using the Agilent scanner (Agilent) at 10 micron and images were analysed using ArrayVision v7 (GE Healthcare).
Description
To study their metabolic potential in natural ecosystems, we developed a species-independent LAB microarray, containing 2,269 30-mer oligonucleotides, and targeting 406 genes that play a key role in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in stress response. Also, genes linked to negative traits such as antibiotic resistance and virulence are represented. This experiment is a validation experiment, where we hybridized labelled RNA from 20 LAB strains, covering 86% of all oligos.
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-ratio for each probe and average the log-ratios over the 4 replicates on the array.
We apply a background correction, i.e. we subtract the local background for the local foreground intensities. We compute the base 2 log-ratio for each probe and average the log-ratios over the 4 replicates on the array.
PRESENT.CALL
the number of times a gene was called present in both channels, i.e. the foreground intensity is above the background intensity plus three times the average of the local standard deviation of the foreground and background pixels. Each clone has four repeats, hence this number varies between 0 and 4.
