Brucella melitensis babR mutant strain was grown with shaking at 37°C in 2YT medium (10% yeast extract, 10 g l-1 tryptone, 5 g l-1 NaCl) from an initial optical density at 600 nm (OD600) of 0.05. Cultures was incubated at 37°C with shaking to an OD600 of 0.75.
Extracted molecule
total RNA
Extraction protocol
Total RNA were extracted from wt, ∆vjbR and ∆babR (all cultured in triplicate) as following: 45 ml of culture (OD600 of 0.75) were centrifuged at 3500 rpm for 15 min. Bacterial pellets were resuspended in 100 µl SDS 10% and 20 µl proteinase K (20 mg ml-1) and incubated at 37°C with shaking for 1h. Five milliliters of TRIzol® Reagent (Invitrogen) were added and suspensions were vigorously shacked. After 10 min of incubation at 65°C, 1 ml chloroform was added to the suspensions and the mixes were shaked and incubated at room temperature for 5-10 min. Samples were centrifuged at 14.000 rpm for 15 min at 4°C. Then, 2.5 ml propan-2-ol were added to the aqueous phases and samples were stored overnight at -20°C. After centrifugation for 30 min at 14.000 rpm at 4°C, pellets were washed with 75% ethanol RNase free. Supernatants were discarded and pellets were dried 15 min at room temperature. Total RNA samples were resuspended in 100 µl RNase free water, incubated 10 min at 55°C and stored at -80°C. The integrity of the RNA and the absence of DNA were checked by gel electrophoresis.
Label
Cy3
Label protocol
1. Prepare the following three solutions: 10X TE (10mM Tris-HCl, 1mM EDTA) 50X dNTP Mix (VWR deionized water 250µl, 10X TE 50µl, 100 mM dGTP 50µl, 100 mM dTTP 50µl, 100 mM dCTP 50µl) Random 9mer Buffer (VWR deionized water 8.6ml, 1M Tris-HCl 1.25ml, 1M MgCl 125µl, 2-Mercaptoethanol 17.5µl)
2. Dilute Cy3 dye-labeled 9mers to 1 O.D./42µl Random 9mer Buffer. Aliquot 40µl in 0.2ml thin-walled PCR tubes and store at -20°C. 3. Assemble the following components in separate 0.2ml thin-walled PCR tubes: cDNA 1µg Cy3-9mer Primers 40µl VWR water To volume Total 80µl 4. Heat-denature samples in a thermocycler at 98C for 10 minutes. Quick-chill in an ice-water bath for 2 minutes. 5. Prepare the following dNTP/Klenow Master Mix:
50X dNTP mix 10µl VWR deionized water 8µl Klenow (50U/µl) 2µl Total 20µl 6. Add the dNTP/Klenow Master Mix to the denatured samples from step 4. Keep on ice.
Reaction volume from step 4 80µl dNTP/Klenow Master Mix 20µl Total 100µl 7. Mix well by pipetting 10 times. 8. Spin down at low RPM to force contents to bottom of tube. 9. Incubate at 37C for 2 hours (3 hours maximum) in a thermocycler protected from light. 10. Stop the reaction by addition of 0.5M EDTA.
Reaction volume from step 6 100µl Stop Solution (0.5M EDTA) 10µl Total 110µl 11. Add 5M NaCl to each tube.
Reaction volume from step 10 110µl 5M NaCl 11.5µl Total 121.5µl 12. Vortex briefly, spin down, and transfer to a 1.5ml tube containing isopropanol.
Reaction volume from step 11 121.5µl Isopropanol 110µl Total 231.5µl 13. Vortex well. Incubate 10 minutes at room temperature, protected from light. 14. Centrifuge at maximum speed of 12,000 x g for 10 minutes. Remove supernatant with pipette. 15. Rinse pellet with 500µl 80% ice-cold ethanol. Dislodge pellet from tube wall. 16. Centrifuge at maximum speed of 12,000 x g for 2 minutes. Remove supernatant with pipette. 17. SpeedVac on low heat for 5 minutes until dry, protected from light. 18. Store labeled samples at -20°C, protected from light. 19. Rehydrate pellets in 25µl VWR deionized water. 20. Vortex for 30 seconds and quick-spin to collect contents at bottom of tube. Continue to vortex or let sit at room temperature, protected from light, until the pellet is completely rehydrated, then vortex again, and quickly spin. 21. Determine the concentration of each sample. The concentration can be determined using the following formula: Concentration (µg/ml) = A260nm x 50 x Dilution Factor 22. Based on the concentration, calculate the amount of sample required for hybridization per the following table and place in 1.5ml tube: Sample Requirements Amount : Prokaryotic sample 1.5µg 23. If you’re ready to perform the hybridization, correct the sample volume to 11.3µl and proceed to Chapter 4, Microarray Hybridization & Washing. a. If the volume is smaller than 11.3µl, bring the volume up to 11.3µl with VWR water. Mix and spin down to collect contents in bottom of tube. b. If the volume is greater than 11.3µl, dry the contents in a SpeedVac on low heat, protected from light. Resuspend the sample in 11.3µl VWR water and vortex to dissolve. Mix and spin down to collect contents in bottom of tube. 24. If you’re not ready to perform the hybridization, dry the contents in a SpeedVac on low heat, protected from light. Store samples at -20C until ready for hybridization.
Hybridization protocol
After labeling, hybridize the sample. Then wash the array before scanning and extracting data. Step 1. Hybridization of Cy-labeled DNA 1. Set MAUI Hybridization System to 42°C. With the cover closed, allow at least 3 hours for the heat block temperature to stabilize at 42°C. 2. If the sample was dried to a pellet at the end of labeling (Chapter 3, step 24 on page 13), resuspend in 11.3µl VWR water. Vortex well and spin down to collect contents in bottom of tube. 3. Using the NimbleGen Hybridization Kit, add the following reagents to the sample:
Hybridization Solution Volume Cy3-labeled sample 11.3µl 2X Hybridization Buffer 19.5µl Hybridization Component A 7.8µl Cy3 CPK6 48mer Oligo, 50nM 0.4µl Total 39.0µl 4. Vortex well (~15 seconds) and then spin to collect the contents in the bottom of the tube. Incubate at 95C for 5 minutes, protected from light. 5. Vortex, spin, and place the tube in the MAUI 42C sample block and incubate at this temperature until you are ready for sample loading. 6. Place the MAUI Mixer SL Hybridization Chamber on the array using the provided assembly/disassembly tool and carefully follow MAUI setup instructions. Use the braying tool to remove all air bubbles from the adhesive gasket around the outside of the hybridization chamber. 7. Place the array and Hybridization Chamber assembly in one of the MAUI bays and allow 30 seconds for the assembly to come to temperature. 8. Load the sample using the pipette supplied with the MAUI Hybridization Station; follow the manufacturer’s instructions. During loading, a small amount of the sample should flow out of the outlet port. 9. Confirm there are no bubbles in the chamber. If there are bubbles, gently massage any bubbles to either end, away from the center of the array. Avoid applying too much pressure because this will force liquid out of the ports. Return the loaded array into one of the MAUI bays. 10. Let equilibrate for 30 seconds. Wipe off any sample leakage at the ports with a clean tissue. Adhere MAUI stickers to both ports, making sure to press on both stickers simultaneously to seal. 11. Close the bay clamp and select mix mode B. Hold down the mix button to start mixing. Confirm that the mixing is in progress before closing the cover. 12. Hybridize the sample overnight (16 to 20 hours).
Scan protocol
This chapter describes the protocol for scanning NimbleGen one-color arrays with a GenePix 4000B Scanner and associated software. 1. Turn on the scanner using the power switch on the back right side. 2. Launch the GenePix software 10 minutes before scanning to allow lasers to warm. 3. Open the scanner door and the slide carriage. 4. Place the array in the slide carriage so that the array is face down and the barcode end is closest to you. 5. Move the black lever on the left side of the carriage to the left until the array is lying flat in the carriage. 6. Release the lever so that the array is gently pushed to the right side of the carriage and held firmly. 7. Close the carriage (you should hear a click) and slide the scanner door shut. 8. Open the Hardware Settings dialog box (right side, second icon from the bottom or Alt + Q). Select the following settings for scanning: Wavelength 532 (Cy3) PMT Gain = 650 Power (%) = 100 Pixel size (µm) = 5 Lines to average = 1 Focus position (µm) = 0 9. Under Image on the left side of the screen, select the 532 wavelength. 10. Perform a preview scan by clicking on the fast-forward icon (or Alt + P). 11. Stop the scan once the full array is in view by pressing the stop icon or the Esc key. 12. Switch to zoom mode by clicking on the array image and pressing Z on the keyboard. Click and drag the cursor over the region in which you want to zoom. To restore the prior view, press Alt + Z. Center the entire array image in the viewing window. 13. Under Tools on the left side of the screen, click the Scan Area icon (or Ctrl + V). Click and drag to define a box that surrounds the array image. The box dimensions should be stretched to create a box just slightly larger than the array image. It is critical to include all of the corner probes within the scan image. 14. Scan the array by clicking the play icon (or Alt + D). While scanning, set the zoom to view the whole image and adjust the brightness and contrast of the displayed image to eliminate visible saturation. 15. Zoom the view to a region scanned under the most recent PMT settings and click on the Histogram tab (top of the left side of the screen) to check global intensity of the features. On the top left side of the screen, make sure the 532 wavelength boxes are checked. On the left side of the screen, under Y-Axis, make sure that the Log Axis box is checked. You want the curve to have 1e-5 normalized counts at the 65,000-intensity level (saturation). This means that you have ~1% to 2% of the features saturated. If the normalized counts at the 65,000-intensity level are less than 1e-5, increase the PMT Gain value (Hardware Settings dialog box). If the normalized counts at the 65,000-intensity level are more than 1e-5, decrease the PMT Gain value (Hardware Settings dialog box). The histogram graphs the region of the image viewable on-screen in the image tab. If the histogram is no longer changing, either the scan is complete or area on the image has been scanned. 16. After the PMT settings are properly adjusted, stop the preview scan and do not save this image. Restart the scan under the new settings. 17. Save the images using the following naming convention:
NNNNN_XXXXX_532.tif
where NNNNN is the barcode number on the slide and XXXXX is user-defined text. 18. After scanning, remove the array from the scanner. Store arrays in a dark desiccator in the event that rescanning is necessary.
Description
Bacterial cultures were made in triplicate. Triplicate RNA samples of the strain were mixed and one chip was made.
Data processing
All of the analysis was performed using the statistical program R (normalisation etc ?), using the package stats (R_Development_Core_Team 2006).
Chip data used were preprocessed using the RMA algorithm (Irizarry, Hobbs et al. 2003), as provided by NimbleGen™ Systems, Inc.
Two pairwise comparisons were performed (∆vjbr-wt) and (∆babr-wt). For each comparison, the foldchange was computed as the ratio of intensity averages (mutant/wt). A Student T test was used as statistical analysis, for over-expression and under-expression.
Genes presenting both a foldchange greater than 1.3 (or below 0.7) and a statistical test significant at the alpha level 0.005 were selected as under/over-expressed between the two conditions.
