genotype/variation: wild type growth condition: filtered RCM until OD.600 of 0.6
Treatment protocol
Cells were collected by centrifugation at 10,000rpm for 2 minutes at room temperature prior to storage in the -80 degree freezer.
Growth protocol
Bifidobacterium breve UCC2003 was grown in filtered reinforced clostridial medium (RCM; Oxoid Ltd.) resuspended in deminerilised water. The RCM media was filtered to remove the agarose to allow cells to be collected by centrifugation this filtration was carried out with the aid of Watt man paper. Iron limtiation was imposed by supplementing the medium with 85µM ciclopirox olamine or 100µM phenanthroline or 275µM Dipyridyl. Bifidobacterial cultures were incubated at 37°C, anaerobically in a modular, atmosphere-controlled system (Davidson and Hardy, Belfast, Ireland) to an optical density OD600 of 0.6.
Extracted molecule
total RNA
Extraction protocol
Method for cell disruption was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).RNA isolation, RNA quality control and complementary DNA synthesis was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).
Label
cy3
Label protocol
Labelling was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).
Channel 2
Source name
B. breve UCC2003 grown in filtered RCM supplemented with 85µM ciclopirox olamine until OD.600 of 0.6
genotype/variation: wild type growth condition: filtered RCM supplemented with 85µM ciclopirox olamine until OD.600 of 0.6
Treatment protocol
Cells were collected by centrifugation at 10,000rpm for 2 minutes at room temperature prior to storage in the -80 degree freezer.
Growth protocol
Bifidobacterium breve UCC2003 was grown in filtered reinforced clostridial medium (RCM; Oxoid Ltd.) resuspended in deminerilised water. The RCM media was filtered to remove the agarose to allow cells to be collected by centrifugation this filtration was carried out with the aid of Watt man paper. Iron limtiation was imposed by supplementing the medium with 85µM ciclopirox olamine or 100µM phenanthroline or 275µM Dipyridyl. Bifidobacterial cultures were incubated at 37°C, anaerobically in a modular, atmosphere-controlled system (Davidson and Hardy, Belfast, Ireland) to an optical density OD600 of 0.6.
Extracted molecule
total RNA
Extraction protocol
Method for cell disruption was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).RNA isolation, RNA quality control and complementary DNA synthesis was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).
Label
cy5
Label protocol
Labelling was performed as described previously (van Hijum, S. A. F. T., J. A. De, R. J. Baerends, H. A. Karsens, N. E. Kramer, R. Larsen, C. D. den Hengst, C. J. Albers, J. Kok & O. P. Kuipers, (2005) A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data. BMC.Genomics 6: 77).
Hybridization protocol
Labelled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050).
Scan protocol
Following hybridization, all microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A).
Description
Gene expression array
Data processing
Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5).
