RNA degradation was stopped using Qiagen's RNAprotect reagent
Growth protocol
Escherichia coli K12 MG1655 was cultivated using M9 minimal medium containing 4.5 g/L α-(D)-glucose, pH = 7, temperature 37 ºC, aerobic conditions
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with Qiagen's Rneasy Mini Kit
Label
Cy5
Label protocol
cDNA was synthesized from 15 μg of total RNA at 46ºC overnight using the following reagents per reaction: 1 μg random primers (Random decamers; Invitrogen, Carlsbad, CA), 6 μL 5 x first strand buffer (Invitrogen), 3 μL 0.1 MDTT (Invitrogen), 0.9 μL dNTPs (final concentrations: dATP 0.5 mM, dCTP 0.5 mM, dGTP 0.5 mM, dTTP 0.3 mM, aminoallyl-dUTP 0.2 mM), 1 μL RNase Inhibitor (Bioron, Ludwigshafen, Germany), 2 μL Superscript III (Invitrogen). RNA strands were hydrolyzed in the samples by adding 4.5 μL 1 M NaOH and incubating at 70ºC for 10 min, after incubation the samples were neutralized with 4.5 μL 1 M HCl. cDNA was purified with MinElute PCR purification kit (QIAGEN) and labeled with Cy3 (CyTM3 Mono Reactive Dye Pack, Amersham, Buckinghamshire, UK) or Cy5 (CyTM5 Mono Reactive Dye Pack, Amersham). Staining was carried out in 10 μL of NaHCO3 (pH 9) at room temperature in dark for 1 h and the samples were purified again with MinElute PCR purification kit.
RNA degradation was stopped using Qiagen's RNAprotect reagent
Growth protocol
Escherichia coli K12 MG1655 was cultivated using M9 minimal medium containing 4.5 g/L α-(D)-glucose, pH = 7, temperature 37 ºC, aerobic conditions
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with Qiagen's Rneasy Mini Kit
Label
Cy3
Label protocol
cDNA was synthesized from 15 μg of total RNA at 46ºC overnight using the following reagents per reaction: 1 μg random primers (Random decamers; Invitrogen, Carlsbad, CA), 6 μL 5 x first strand buffer (Invitrogen), 3 μL 0.1 MDTT (Invitrogen), 0.9 μL dNTPs (final concentrations: dATP 0.5 mM, dCTP 0.5 mM, dGTP 0.5 mM, dTTP 0.3 mM, aminoallyl-dUTP 0.2 mM), 1 μL RNase Inhibitor (Bioron, Ludwigshafen, Germany), 2 μL Superscript III (Invitrogen). RNA strands were hydrolyzed in the samples by adding 4.5 μL 1 M NaOH and incubating at 70ºC for 10 min, after incubation the samples were neutralized with 4.5 μL 1 M HCl. cDNA was purified with MinElute PCR purification kit (QIAGEN) and labeled with Cy3 (CyTM3 Mono Reactive Dye Pack, Amersham, Buckinghamshire, UK) or Cy5 (CyTM5 Mono Reactive Dye Pack, Amersham). Staining was carried out in 10 μL of NaHCO3 (pH 9) at room temperature in dark for 1 h and the samples were purified again with MinElute PCR purification kit.
Agilent Technologies Microarray Scanner G2565CA was used at 5 mm resolution
Description
Investigation of specific growth rate dependent gene expression changes of E. coli growing on glucose minimal medium
Data processing
Image was analized with Genepix 6 pro followed by background substration and global LOWESS normalization in R environment (KTH package). Spots with signal to noise ratio lower that 3 and/or relative standard deviation between parallel spots greater than 20% were removed.
Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase
Data table header descriptions
ID_REF
VALUE
normalized log2 ratio (Cy5/Cy3) representing test/reference
