5 protocols
Just before hybridizations, microarrays slides were blocked as following. Slides were successively immersed 5 min at room temperature in a Triton X100 aqueous solution (0.1 %), 2 x 2 min at room temperature in a HCl aqueous solution (0.0037%), 10 min at room temperature in a HCl aqueous solution (0.0037%), 10 min at room temperature in a KCl aqueous solution (0.1M), 1 min at room temperature in water, 20 min at 50 C in an aqueous blocking solution (HCl 0.0074%, QMT 1X (Interchim)), and 1 min at room temperature in water. Then the slides were dried by centrifugation. Hybridizations were conducted using a Lucidea automated slide processor (Amersham Pharmacia Biotech) under the following conditions. Microarrays slides were hybridized at 42 C for 15 hours in DIG Easy hybridization solution (Roche). Following hybridization, slides were washed in 2X SSC-0.2% SDS for 5 min at 42 C, 2 X in 0.2X SSC-0.1%SDS for 1 min at 25 C, 2 x in 0.2X SSC for 1 min at 25 C, and in 0.1X SSC for 1 m in at 25 C. Then the slides were washed with isopropanol and dried.
The protocol is according to Ruberg et al. 2003.J. Biotechnol. 106:255-268. Total RNA was first converted to cDNAs in the presence of aminoallyl-dUTP using Superscript II reverse transcriptase (Invitrogen). Twenty µg of total RNA was combined with 6 µg of random hexamers (Invitrogen) and incubated at 70 C for 10 min. After rapid cooling at 4 C for 30 sec, the reaction mixture was brought to a final volume of 30 µl by adding 6 µl of 5X Superscript II reverse transcriptase buffer, 3 µl of 0.1 mM dithiothreitol (DTT), 0.5 µl of RNasin, 0.6 ul of a 50X dNTP-ammino allyl dUTP mixture (0.5 M each of dGTP, dATP and dCTP, 0.16 mM dTTP, 0.34 mM aminoallyl-dUTP) and 1.5 µl (200 units/µl) of Superscript II reverse transcriptase. After incubation at 42 C for 1 hour, 1 µl of Superscript II reverse transcriptase was added and the mixture was incubated for 1 more hour at 42 C. After completion of the first strand synthesis, the RNA was hydrolyzed by adding 15 µl of 0.2 N NaOH and incubatio n for 10 min at 70 C. Neutralization was performed by adding 15 µl of 0.2 N HCl. The aminoallyl-cDNA was purified from free amines and unincorporated aminoallyl-dUTP using Qiaquick PCR spin columns (Qiagen). After elution, the aminoallyl-cDNA was dried under vacuum in a SpeedVac and resuspended in 5 µl of 0.1 M sodium bicarbonate (pH 9.0). For fluorochrome labeling, 5 µl of a solution of fluoroLink Cy3 or Cy5 dye (Amersham), prepared by resuspending one reaction vial into 45 µl of DMSO, were added and incubated for 1 h at room temperature in the dark. This reaction was quenched by adding 5 µl of 4M hydroxylamine and incubated for 15 min at room temperature in the dark. Fluorescent labeled cDNAs were purified using Qiaquick PCR spin columns and eluted 2 times in 30 µl of manufacturer elution buffer.
S. meliloti 1021 cells were grown in 150 ml GTS liquid medium (2 g/l glucose, 2.7 g/l sodium succinate, 3 g/l Tris-HCl, 2 g/l (NH4)2SO4, 1 g/l NaCl, 0.1 g/l K2HPO4, 246 mg/l MgSO4 x 7 H2O, 11 mg/l CaCl2, 0.27 mg/l FeCL3 x 6 H2O, 0.242 mg/l Na2MoO4 x 2 H2O, 3 mg/l H3BO3, 2.23 mg/l MnSO4 x 4 H2O, 0.287 mg/l ZnSO4 x 7 H2O, 0.125 mg/l CuSO4 x 5 H2O, 0.065 mg/l CoCl2, 2 mg/l biotin) for 10 hours to an OD600 of 0.2 - 0.3 on a shaking incubator (28 C, 200 rpm). Cells were divided into 3 cultures of 50 ml; one culture as control, one culture was treated with 50 micromolar cadmium chloride, and one culture with 100 micromolar zinc sulfate. All three cultures were incubated for 2 hours with further shaking at 30 C. Cells were harvested by filtration in 25 ml aliquots, immediately frozen in liquid nitrogen and stored at -80 C until RNA extraction.
The bacterial cells were resuspended from the filter by vortexing several times during incubation for 20 min at 65 C in 3 ml of prewarmed lysis solution (1.4% SDS, 4mM EDTA, 0.4 mg/ml of proteinase K). Proteins were precipitated by adding 1.5 ml of 5 M NaCl and incubation for 10 minutes on ice. Nucleic acids were precipitated from the supernatant by addition of 2 volumes of ethanol and the pellet was resuspended in 100 µl of RNAse free water. RNA samples were then treated with DNaseI using the Qiagen RNeasy clean-up procedure.
Hybridized slides were scanned using GenePix 4000 dual-channel (635nm and 532nm) confocal laser scanner with a resolution of 10nm per pixel. Laser power was set at 100 and photomultiplicator tension (PMT) was adjusted between 630V and 850V according to the average intensity of the hybridization of each slide in order to optimize the dynamic range of measurements. Quantification of the signals from individual slides was done using GenePix Pro version 3.0 (Axon) software and analyzed using Genesight version 3.5 (Biodiscovery Inc.).