Inoculum cultures were obtained by inoculating a few colonies in 10 ml LB broth and incubated statically at 25°C for 24 hr. Then a second subculture was made by inoculation 100 µl of the first subculture in 10 ml LB broth, which was incubated under the same conditions. For immobilised and planktonic growth of Salmonella was the IFR Gel Cassette system used. For immobilised growth, a 24 hr standard inoculum culture was diluted 1000-fold in diluents, which was futher diluted 1000-fold in LB with 22% (w/w) Pluronic® to give 103 CFU g per gel. This mixture was loaded into sterile Gel Cassettes. For planktonic growth in the Gel Cassette system, metal holders with a metal mesh or acetal frames with 644 holes was used. Similar to the gel, the standard inocula were diluted and inoculated at 103 CFU per ml in LB broth. Immobilised and planktonic cultures were incubated at 25°C without aeration.
Extracted molecule
total RNA
Extraction protocol
1) Harvest 2.0 OD600 units of bacterial culture (for example 4 mls of a culture with an OD600 = 0.5) 2) Prepare a 50 ml Falcon tube containing 2/5 of the culture volume ice-cold 5% (v/v) phenol pH 4.3, 95% (v/v) ethanol (Phenol Sigma order code: P-4682). Add the appropriate volume of culture e.g. add 4 mls of culture to 1.6 mls of phenol/ethanol. Stand on ice for at least 30 min but no longer than 2 hours to stabilise the RNA and prevent degradation. 3) Centrifuge samples at 3220 X g 4°C for 10 min. Discard supernatant, resuspend bacterial pellets using residual liquid in tubes and transfer to 1.5 ml microcentrifuge tubes. 4) Spin tubes 60 secs at maximum speed in a microfuge and discard remaining liquid. 5) Optional: Freeze pellets at -80°C. Pellets can be kept for up to 1 month before continuing with RNA prep. 6) Resuspend pellets in 100 μl TE buffer containing 50 mg/ml lysozyme. Incubate at room temperature for 5 min. 7) Add 75 μl lysis reagent (Promega SV Total RNA Purification kit. Cat: Z3100) and mix by inversion several times. 8) Add 350 μl RNA dilution buffer from kit (Promega SV, Cat: Z3100). Mix well by inversion. 9) Heat samples at 70 °C for 3 min and then centrifuge for 10 min at full speed (13000 rpm). 10) Transfer supernatant to clean tubes supplied with the kit. Add 200 μl ethanol and mix by pipette. Transfer to spin columns (Cat: Z3100) and centrifuge columns for 30 secs at full speed. Discard eluate. 11) Wash columns with 600 μl wash buffer from the kit. Spin 30 secs at full speed. 12) Prepare DNase mix (all reagents supplied with the kit): a) 5 μl 90 mM MnCl2 b) 40 μl DNase core buffer c) 5 μl DNase 13) Apply 50 μl of DNase mix to column matrix and incubate at room temperature for 15 min. 14) Add 200 μl DNase stop mix (kit) and centrifuge 30 secs at full speed. 15) Wash columns with 600 μl wash buffer, by centrifugation for 30 secs at full speed. Discard eluate. 16) Wash columns with 250 μl wash buffer, by centrifugation for 30 secs at full speed. Discard eluate. 17) Transfer columns to sterile microcentrifuge tubes, add 100 μl RNAse-free distilled H2O and allow to stand for 1 min. 18) Centrifuge at 4500 X g for 2 min. Discard column. 19) Check the RNA concentration using a spectrophotometer. From exponential phase cultures, you should typically obtain 50-60 μg RNA. Stationary phase and near-stationary phase samples will yield less, but nevertheless plenty to use. Quality of RNA should also be checked by the Aligent Bioanalyzer or a similar method to ensure the RNA is not degraded.
Label
Cy5
Label protocol
Set up random priming reactions in 1.5 ml microfuge tubes. Add: a) 10 μg of total RNA (this may require concentrating using a speed vac) b) 5 μg of random hexamers (Invitrogen, Cat: 48190011) c) In a total volume of 9.4 μl of Sigma ultra-pure water (mol. biol. reagent, Cat: W4502). d) Incubate 70°C for 5 min protected from light then chill on ice for 10 min. Spin briefly in microfuge. 2) Using the Stratagene AffinityScript multi-temperature Reverse Transcriptase (Cat: 600109) Prepare RT reaction mix (sufficient for one labelling reaction) a) 2.0 μl of 10 X RT buffer b) 2.0 μl of 0.1 M DTT c) 0.6 μl of 50 X dNTP's * 3) Add RT reaction mix to RNA (4.6 μl per reaction) 4) Finally add: a) 2 μl of Cy3 or Cy5-dCTP (1mM stock, GE Healthcare Lifesciences, Cat: PA55321) b) 4 μl of reverse transcriptase. The total reaction volume is 20 μl. 5) Mix and incubate at 25oC for 10 mins. 6) Incubate overnight at 42°C. 7) Add 15 μl of freshly prepared 0.1M NaOH and hydrolyse the RNA at 70°C for 10 minutes. Add 15 μl of 0.1M HCl to neutralise the alkali. 8) If performing a type I experiment follow section ‘a’ or for a type II follow section ‘b’ a) If performing a type I experiment (RNA verses RNA) (DeRisi et al, Science, 1997, 278, 680-686), combine reactions and clean up using Qia-quick PCR purification kit (Qiagen, Cat: 28104) to remove unincorporated/quenched cy dyes. Elute twice using 50 μl Sigma water as final eluant to maximise recovery. Speed vac on medium setting and redissolve pellet in 10 μl Sigma water (mol. biol. reagent, Cat: W4502). Proceed to 'Microarray Hybridisations' protocol. b) If performing a type II experiment (RNA verses genomic DNA), mix labelled cDNA with labelled genomic DNA (see 'Direct labelling of DNA' protocol). Clean up using Qia-quick PCR purification kit (Qiagen, Cat: 28104) to remove unincorporated/quenched cy dyes. Elute twice using 50 μl Sigma water as final eluant to maximise recovery. Speed vac on medium setting and redissolve pellet in www.ifr.ac.uk/safety/molmicro Molecular Microbiology Group, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA UK 10 μl Sigma water (mol. biol. reagent, Cat: W4502). Proceed to 'Microarray Hybridisations' protocol. *To prepare 50 X dNTP's, mix 25 mM dATP, dGTP, dTTP and 10 mM dCTP, (25 μl of dA, dG, dT and 10 μl of dCTP from 100 mM stock dNTP kit (GE Healthcare Lifesciences, Cat: 27-2035-02). Add Sigma water to 100 μl. NB: Sigma water (molecular biology reagent, Cat: W4502) was used to prepare all solutions
strain: ST4/74 sample type: genomic DNA as reference
Growth protocol
Inoculum cultures were obtained by inoculating a few colonies in 10 ml LB broth and incubated statically at 25°C for 24 hr. Then a second subculture was made by inoculation 100 µl of the first subculture in 10 ml LB broth, which was incubated under the same conditions. For immobilised and planktonic growth of Salmonella was the IFR Gel Cassette system used. For immobilised growth, a 24 hr standard inoculum culture was diluted 1000-fold in diluents, which was futher diluted 1000-fold in LB with 22% (w/w) Pluronic® to give 103 CFU g per gel. This mixture was loaded into sterile Gel Cassettes. For planktonic growth in the Gel Cassette system, metal holders with a metal mesh or acetal frames with 644 holes was used. Similar to the gel, the standard inocula were diluted and inoculated at 103 CFU per ml in LB broth. Immobilised and planktonic cultures were incubated at 25°C without aeration.
Extracted molecule
genomic DNA
Extraction protocol
1) Harvest 2.0 OD600 units of bacterial culture (for example 4 mls of a culture with an OD600 = 0.5) 2) Prepare a 50 ml Falcon tube containing 2/5 of the culture volume ice-cold 5% (v/v) phenol pH 4.3, 95% (v/v) ethanol (Phenol Sigma order code: P-4682). Add the appropriate volume of culture e.g. add 4 mls of culture to 1.6 mls of phenol/ethanol. Stand on ice for at least 30 min but no longer than 2 hours to stabilise the RNA and prevent degradation. 3) Centrifuge samples at 3220 X g 4°C for 10 min. Discard supernatant, resuspend bacterial pellets using residual liquid in tubes and transfer to 1.5 ml microcentrifuge tubes. 4) Spin tubes 60 secs at maximum speed in a microfuge and discard remaining liquid. 5) Optional: Freeze pellets at -80°C. Pellets can be kept for up to 1 month before continuing with RNA prep. 6) Resuspend pellets in 100 μl TE buffer containing 50 mg/ml lysozyme. Incubate at room temperature for 5 min. 7) Add 75 μl lysis reagent (Promega SV Total RNA Purification kit. Cat: Z3100) and mix by inversion several times. 8) Add 350 μl RNA dilution buffer from kit (Promega SV, Cat: Z3100). Mix well by inversion. 9) Heat samples at 70 °C for 3 min and then centrifuge for 10 min at full speed (13000 rpm). 10) Transfer supernatant to clean tubes supplied with the kit. Add 200 μl ethanol and mix by pipette. Transfer to spin columns (Cat: Z3100) and centrifuge columns for 30 secs at full speed. Discard eluate. 11) Wash columns with 600 μl wash buffer from the kit. Spin 30 secs at full speed. 12) Prepare DNase mix (all reagents supplied with the kit): a) 5 μl 90 mM MnCl2 b) 40 μl DNase core buffer c) 5 μl DNase 13) Apply 50 μl of DNase mix to column matrix and incubate at room temperature for 15 min. 14) Add 200 μl DNase stop mix (kit) and centrifuge 30 secs at full speed. 15) Wash columns with 600 μl wash buffer, by centrifugation for 30 secs at full speed. Discard eluate. 16) Wash columns with 250 μl wash buffer, by centrifugation for 30 secs at full speed. Discard eluate. 17) Transfer columns to sterile microcentrifuge tubes, add 100 μl RNAse-free distilled H2O and allow to stand for 1 min. 18) Centrifuge at 4500 X g for 2 min. Discard column. 19) Check the RNA concentration using a spectrophotometer. From exponential phase cultures, you should typically obtain 50-60 μg RNA. Stationary phase and near-stationary phase samples will yield less, but nevertheless plenty to use. Quality of RNA should also be checked by the Aligent Bioanalyzer or a similar method to ensure the RNA is not degraded.
Label
Cy3
Label protocol
Set up random priming reactions in 1.5 ml microfuge tubes. Add: a) 10 μg of total RNA (this may require concentrating using a speed vac) b) 5 μg of random hexamers (Invitrogen, Cat: 48190011) c) In a total volume of 9.4 μl of Sigma ultra-pure water (mol. biol. reagent, Cat: W4502). d) Incubate 70°C for 5 min protected from light then chill on ice for 10 min. Spin briefly in microfuge. 2) Using the Stratagene AffinityScript multi-temperature Reverse Transcriptase (Cat: 600109) Prepare RT reaction mix (sufficient for one labelling reaction) a) 2.0 μl of 10 X RT buffer b) 2.0 μl of 0.1 M DTT c) 0.6 μl of 50 X dNTP's * 3) Add RT reaction mix to RNA (4.6 μl per reaction) 4) Finally add: a) 2 μl of Cy3 or Cy5-dCTP (1mM stock, GE Healthcare Lifesciences, Cat: PA55321) b) 4 μl of reverse transcriptase. The total reaction volume is 20 μl. 5) Mix and incubate at 25oC for 10 mins. 6) Incubate overnight at 42°C. 7) Add 15 μl of freshly prepared 0.1M NaOH and hydrolyse the RNA at 70°C for 10 minutes. Add 15 μl of 0.1M HCl to neutralise the alkali. 8) If performing a type I experiment follow section ‘a’ or for a type II follow section ‘b’ a) If performing a type I experiment (RNA verses RNA) (DeRisi et al, Science, 1997, 278, 680-686), combine reactions and clean up using Qia-quick PCR purification kit (Qiagen, Cat: 28104) to remove unincorporated/quenched cy dyes. Elute twice using 50 μl Sigma water as final eluant to maximise recovery. Speed vac on medium setting and redissolve pellet in 10 μl Sigma water (mol. biol. reagent, Cat: W4502). Proceed to 'Microarray Hybridisations' protocol. b) If performing a type II experiment (RNA verses genomic DNA), mix labelled cDNA with labelled genomic DNA (see 'Direct labelling of DNA' protocol). Clean up using Qia-quick PCR purification kit (Qiagen, Cat: 28104) to remove unincorporated/quenched cy dyes. Elute twice using 50 μl Sigma water as final eluant to maximise recovery. Speed vac on medium setting and redissolve pellet in www.ifr.ac.uk/safety/molmicro Molecular Microbiology Group, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA UK 10 μl Sigma water (mol. biol. reagent, Cat: W4502). Proceed to 'Microarray Hybridisations' protocol. *To prepare 50 X dNTP's, mix 25 mM dATP, dGTP, dTTP and 10 mM dCTP, (25 μl of dA, dG, dT and 10 μl of dCTP from 100 mM stock dNTP kit (GE Healthcare Lifesciences, Cat: 27-2035-02). Add Sigma water to 100 μl. NB: Sigma water (molecular biology reagent, Cat: W4502) was used to prepare all solutions
Hybridization protocol
Slides blocked with DCE according to protocol at PMID 11266573. Microarray Hybridisations 1. To 10 μl of dried down labelling reactions add: a) 1.5 μl of 50 X Denhardts solution b) 2.25 μl of 20 X SSC c) 1.125 μl of E. coli tRNA (10 μg/μl) (Sigma, Cat: R8759) d) 0.375 μl of 1M HEPES, pH 7.0. NOTE: The hybridisation components can be mixed together as a stock solution, either from a fresh or frozen stock, and added to labelling reactions. 2. Add 0.375 μl of 10% SDS to the mixture 3. Incubate 100 °C for 2 min. Let stand on the bench for 5 - 10 min. Do not place the reaction on ice because the SDS will precipitate. 4. Spin in a microfuge at full speed for 5 min, transfer supernatant to clean tube and repeat spin. 5. Place a microarray slide in a metal hybridisation chamber (Photo 3). 6. Pipette the hybridisation solution towards one edge of an array. 7. Place edge of a clean (washed with 70% ethanol), dry, coverslip on the edge of the array. 8. Position fine nosed forceps (VWR, Cat: 406/0073/04) under the coverslip. 9. Using the forceps slowly lower the coverslip down onto the hybridisation solution and the array ensuring no air bubbles are present under the coverslip. If air bubbles are present it is sometimes possible to move them to the edge of the coverslip by pressing gently with the forceps. 10. Apply 20 μl of 3 X SSC around the 4 corners of the slide using a pipette (approximately 5 μl per corner). This will maintain the correct humidity in the hybridisation chamber. 11. Screw the lid onto the chamber and place at the bottom of a water bath at 63°C overnight. 12. Remove the slides from the hybridisation chambers and place them in a plastic slide rack (Photo 4). The slides are then placed into a 63°C wash solution containing 2 X SSC, 0.1% SDS and agitated for 5 mins. (We use a 2L beaker with a stirrer bar in the bottom and a platform for the slide rack to sit on for this, Photos 5 & 6). It may be necessary to tap the slide rack on the platform to make sure that the cover slips are removed. We use a thermocouple attached to a hot-plate stirrer for this step. Repeat the washing step. Note: SDS fluoresces so you should avoid carrying it over into the washes in steps 13 and 14. 13. Transfer slide rack to a solution of 1 X SSC at room temperature and agitate for 5 min. Repeat. (We use black tissue boxes often used for histology for this) 14. Wash in 0.2 X SSC for 5 min. Repeat. 15. Spin dry in a centrifuge at 1200 rpm for 5 min at room temp in an enclosed slide chamber (with blotting paper lining the bottom of the chamber). 16. Scan. NB All solutions are filter sterilised (0.1 μm) and made up with Sigma water (molecular biology reagent, Sigma, Cat: W4502).
Intensity ratios of channel 1 (cDNA) divided by channel 2 (reference gDNA) signals are calculated using BlueFuse 3.1 or 3.6 (BlueGnome, Cambridge, UK). Data centering was subsequently performed using the block median
