B. subtilis strains were grown in TY medium (1% tryptone, 0.5% yeast extract and 1% NaCl) at 37°C, 250 rpm containing the appropriate antibiotic: spectinomycin (100 μg/ml).
Extracted molecule
total RNA
Extraction protocol
RNA was isolated by spinning down 2 ml of culture (30 s Eppendorf centrifuge, 14 000 r.p.m., 4°C) and resuspending the pellet in 0.3 ml ice-cold growth medium (final volume 0.4 ml). The cell suspension was added to a screw cap Eppendorf tube containing 1.5 g glass beads (75±150 mm), 0.5 ml phenol:chloroform:isoamylalcohol (12:12:1), 50 μl 10% SDS and 50 μl 3M sodium acetate (pH 5.2). All solutions were prepared with diethylpyrocarbonate (DEPC)-treated water. After vortexing, the tube was frozen in liquid nitrogen and stored at ±80°C. Cells were broken by shaking for 8 min in a shake-it-baby (15). After 5 min centrifugation (Eppendorf centrifuge, 10 000 r.p.m., 4°C) the water phase (0.4 ml) was transferred to a clean tube containing 0.4 ml chloroform. After vortexing and centrifugation (2 min, Eppendorf centrifuge, 14 000 r.p.m., 4°C), the water phase was transferred to a clean tube and the RNA was isolated with a High Pure RNA Isolation Kit (Roche). RNA was eluted in 50 μl elution buffer and quanti®ed with GeneQuant (Amersham).
Label
33P
Label protocol
Reverse transcription was carried out in a total volume of 50 μl. Aliquots of 1 pmol of B.subtilis ORF-speci®c primers (Eurogentec) were mixed with 4 μg isolated RNA, incubated for 10 min at 70°C and cooled on ice. Subsequently, 10 μl of 5x concentrated First strand buffer (Gibco BRL), 5 vl 100mM DTT, 0.5 μl RNasin (Roche, 40 U/ml), 2.5 μl dNTP mixture (5 mM dATP, 5 mM dTTP, 5 mM dGTP, 0.1 mM dCTP) were added, and the total volume was adjusted to 42.5 μl with DEPC-treated water, and kept on ice. After addition of 5 ml [alfa-33P]dCTP, the mixture was incubated for 10 min at 25°C, before the addition of 2.5 ml SuperscriptII (Gibco BRL). Reverse transcription reaction was carried out for 2 h at 42°C, 15 min at 70°C, and was stopped by adding 2 μl 0.5 M EDTA, 2 μl 10% SDS, 6 μl 3 M NaOH and incubating for 30 min at 68°C. After neutralization with 6 μl 3 M HCl, the labeled cDNA was puri®ed on a Sephadex G-25 column (Roche). Incorporation of label was checked by scintillation counting.
Hybridization protocol
Panorama hybridisation solution (PRHY0001) by Sigma was used for hybridisation as described by the manufacturer, ie: 1. Rinse the blots in 50 ml 2x SSPE for 5 minutes. Drain and discard the solution. 2. Pre-warm the hybridization oven to 65°C. Warm the Hybridization Solution (Sigma-Genosys Cat. No. PRHY0001 or Kit Cat. No. PRLB0001) to 65°C for about 10 minutes prior to use. Add the appropriate quantity of salmon testes DNA to achieve a final concentration of 100 μg/ml, immediately prior to using the Hybridization Solution. 3. Pre-hybridize the Panorama gene array in 5 ml Hybridization Solution for at least 1 hour at 65°C, using roller bottles at 6 r.p.m. (or continuously agitate if using sealed bags). 4. Add the entire labeled cDNA generated from Part Two of this protocol to 3 ml Hybridization Solution. Heat at 90 – 95°C for 10 minutes in a water bath to denature the cDNA. 5. Decant and discard the Hybridization Solution from the pre-hybridized array. Add the denatured labeled cDNA in Hybridization Solution to the array in the roller bottle. 6. Hybridize overnight (12 – 18 hours) at 65°C. 7. Decant the hybridization solution and save for future use or discard appropriately. 8. Add 40 – 50 ml of Wash Solution to the roller bottle. Wash the array by inverting the roller bottle by-hand, at room temperature for 2 – 3 minutes. Decant and discard the Wash Solution in an appropriate manner for radioactive waste solutions. Note: An alternative wash method may be adopted for the arrays. Arrays may be washed in a suitably-sized plastic food container (dedicated for use with radioactive materials). Agitate the container on a rocking table or use a shaking water bath. 9. Repeat step 8 two more times. 10. Pre-warm the Wash Solution to 65°C. Add 80 – 100 ml Wash Solution to the roller bottle. Wash filters in the hybridization oven at 65°C for 20 minutes (6 r.p.m.). Decant and discard the Wash Solution to an appropriate radioactive waste container. 11. Repeat step 10 two more times. 12. Remove the array from the roller bottle (or the alternative washing container). Lay the array on a sheet of blotting paper. 13. Air-dry the array for 2 – 3 minutes. 14. Wrap the array in clear food wrap and subject to autoradiography using Cyclone phospho-imager screens (Packard) for ~2.5 days
Scan protocol
After hybridization and washing, Cyclone phospho-imager screens (Packard) were exposed for ~2 days and read out with the Cyclone at 300dpi.
Description
none
Data processing
Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel. Outstandingly high signals (for example whole genomic DNA control spots which act as a positive control and always show very strong signals) were excluded from calculations of the total signal. Statistical significance of the obtained gene expression ratios was assessed by the Cyber-T program
