Zn-depleted custom-built chemostats were grown for 50 h. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. The culture was pipetted directly into RNAprotect (Qiagen) to stabilize RNA. Total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. RNA was quantified using a BioPhotometer (Eppendorf). A control experiment was carried out in which water was added.
Label
Cy3
Label protocol
RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC. This was supplemented with 4 microlitres of 5× First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres l 0.1 M DTT, 2 microlitres 1 mM Cy3 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1 M) and HCl (5 microlitres of 1 M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick purification kit (Qiagen).
Zn-depleted custom-built chemostats were grown for 50 h. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. The culture was pipetted directly into RNAprotect (Qiagen) to stabilize RNA. Total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. RNA was quantified using a BioPhotometer (Eppendorf). A control experiment was carried out in which water was added.
Label
Cy5
Label protocol
RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC. This was supplemented with 4 microlitres of 5× First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres l 0.1 M DTT, 2 microlitres 1 mM Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1 M) and HCl (5 microlitres of 1 M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick purification kit (Qiagen).
Hybridization protocol
The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 1× SSC/0.1 % SDS, 1× SSC, 0.2× SSC and 0.01× SSC. Microarray slides were dried by centrifugation at 500 × g for 2 min before scanning.
Scan protocol
Slides were scanned on an Affymetrix 428 scanner.
Data processing
The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.
