Standard BuG@S ImaGene upload protocol. Parameters are read from the ImaGene file during the upload process.
Excess Cy3 and Cy5 dCTP was removed from labelled DNA or cDNA samples using the MinElute Reaction Cleanup Kit (Qiagen). Cy3 and Cy5 labelled DNA and/or cDNA samples were combined in a single tube (1.5mL) and 5 volumes of PB buffer added. The solution was applied to a MinElute column in a collection tube and centrifuged at 13,000rpm for 1min. The flow-through was discarded and the MinElute column placed back into the same collection tube. 500uL of Buffer PE was added to MinElute column, centrifuged at 13,000rpm for 1min and the flow-through discarded. The MinElute column was placed back in the same collection tube and the previous step repeated with 250uL of PE. The MinElute column was placed into a fresh 1.5mL tube, 15.9uL of water (22x22mm LifterSlip) or 30.2uL (22x50mm LifterSlip) was added to the centre of the membrane, allowed to stand for 1min and then centrifuged at 13,000rpm for 1min. 50mL of prehybridization solution (3.5xSSC, 0.1% SDS, 10mg/mL BSA) was placed in Coplin jar and incubated at 65C to preheat for 1h 30min. The microarray slide was placed in the pre-hybridization solution and incubated at 65C for 20min. The slide was then rinsed in 400mL water for 1min and 400mL of propan-2-ol for 1min. The slide was placed in a 50mL centrifuge tube and centrifuged at 1,500rpm for 5min to dry. Each slide was stored in a dark, dust free box until hybridization (<1h). The prehybridized microarray slide was placed in the hybridization cassette and two 15uL aliquots of water added to the wells of the cassette. A 4xSSC 0.3% SDS hybridization solution containing the Cy3/Cy5 labelled samples was prepared with a final volume of 23ul (22x22mm LifterSlips)or 45uL (22x50mm LifterSlips). The hybridization solution was heated at 95C for 2min, allowed to cool slightly at room temperature and briefly centrifuged. A LifterSlip was placed carefully over the arrayed area of the slide, to avoid scratching its surface. The hybridization solution was pipetted under one corner of the LifterSlip, allowing the solution to be drawn completely across the array by capillary action. Any excess hybridization solution was pipetted under the opposite corner of the LifterSlip. The hybridization cassette was sealed and submerged in a water bath at 65C in the dark for 16-20h. Wash A (1xSSC, 0.05% SDS) was preheated to 65C and placed in a staining trough pre-heated to 65C. The microarray slide was removed from the hybridization cassette and carefully washed in the staining trough of Wash A at 65C to remove LifterSlip. The slide was then placed in a slide rack and agitated in Wash A for a further 2min. Slides were transferred to a clean rack and agitated in a trough of 400mL of Wash B (0.06xSSC) for 2min at room temperature. Slides were transferred to a second trough of 400mL of Wash B (0.06xSSC) and agitated for a further 2min at room temperature. The slide was then dried by centrifugation at 1,500rpm for 5min in a 50mL centrifuge tube.
1ug of each DNA sample was placed in a microfuge tube (0.5mL) with 3ug of random primers (1uL) made up to a final volume of 42.5uL with H2O (DNase and RNase free, molecular biology grade). The DNA was then heated to 95C, snap-cooled on ice and centrifuged. 5uL REact buffer 2 (x10), 1uL dNTP's (5mM dA/G/TTP, 2mM dCTP), 1.5uL of Cy3 dCTP (1mM) and 1uL of Klenow (3-9U/uL) were added to make a final volume of 50uL. The solution was incubated in the dark at 37C for 90min.
Dye reversals were performed. Spots with Flags of -50 were removed from analyses. To be included in the table, the log ratio (Alexa Fluor 647/Alexa Fluor 555) was greater than 1.5/less than -1.5 for two slides (at least 3 slides for the WCH206 and Menzies17 comparison).
Data Transformation - Set measurements Values below 0.01 were set to 0.01. Per Spot Normalization - Divide by control channel (Per Spot Cutoff 0.01) Each genes measured intensity was divided by its control channel value in each sample. Per Chip Normalization - Normalize to 50th percentile (Per Chip Flag Restrict Present or Marginal). Each measurement was divided by the 50th percentile of all measurements in that sample.
Raw data files from ImaGene generated for multiple scans at a range of PMT gain settings were averaged by MAVI Pro software to improve the dynamic range of data and also account for low intensity and saturation effects. The folder of multiple ImaGene text files for each array were processed by MAVI Pro and output in ImaGene format.
2-10ug of RNA sample was placed in a microfuge tube (0.5mL) with 3ug of random primers (1uL) and made up to a final volume of 11uL with H2O (DNase and RNase free, molecular biology grade). The RNA was then heated to 95C for 5min, snap cooled on ice and centrifuged. 5uL First Strand Buffer (5x), 2.5uL DTT (100mM), 2.3ul dNTP's (5mM dA/G/TTP, 2mM dCTP), 1.7uL of Cy5(1mM) and 2.5uL of SuperScript II (200U/uL) were added to make a final volume of 25uL. The solution was incubated in the dark at 25C (room temperature) for 10min and then at 42C in the dark for 90min.
Mycobacterial Retrieval 4vol of 5M GTC solution were added to the mycobacterial liquid culture and mixed (GTC solution - 5M guanidine thiocyanate, 0.5% sodium N-lauroyl sarcosine, 25mM sodium citrate, 1%Tween-80, 0.1MB-mercaptoethanol, see below for preparation details). 25mL aliquots of the culture:GTC mixture were then centrifuged in 30mL universal tubes at 3,000rpm for 30min. Universal and not falcon tubes should be used as the bacilli centrifuge into tighter pellets using universals. Mycobacteria should not lyse on addition of GTC solution, an increase in viscosity on addition of GTC solution may indicate bacterial or eukaryotic cell contamination. Mycobacterial transcription ceases on addition of GTC solution, however do not centrifuge bacterial cultures or place on ice etc before addition of GTC solution as the transcription profile of bacilli will change. The supernatant was removed and the bacterial pellets re-suspended in 1mL spent GTC for every 50mL of original culture volume (a higher ratio was used for stationary phase cultures or cultures where the bacteria were clumped).1mL aliquots of resuspended bacteria were transferred into 2mL skirted apex tubes (ribolyser tubes), centrifuged for 30s at 13,000rpm and the excess GTC removed from the bacterial pellets. If RNA extraction is to be completed elsewhere, add 1mL Tri Reagent (Sigma) to each bacterial pellet, mix and store at -80C. Transport as hazardous substance on dry ice. Otherwise this is not a recommended stopping point. Nucleic Acid Extraction 1.2mL Tri Reagent (Sigma) and 0.5mL of 0.1mm silica beads (Lysing matrix B, Q Biogene; or contents of a blue ribolyser tube from Hybaid) were added to the bacterial pellet in each tube and mixed briefly (using approximately 1 ribolyser tube/50mL mycobacterial culture). The bacterial cells resuspended in Tri Reagent were lysed using a reciprocal shaker (FastPrep FP120, AB Gene, Thermo Savant; or Hybaid ribolyser) for 45s at a speed of 6.5. Disrupted cells were then left at room temperature for 10min. It is now safe to remove preparations of pathogenic mycobacteria from Category Three containment conditions. 200µL chloroform was then added to each cell suspension, vortexed for 30s, and incubated at room temperature for a further 10min to partition the aqueous and phenolic phases before centrifuging at 13,000rpm for 15min at 4C. On addition of chloroform the pink colouring of the tri-reagent should remain in the lower organic phase, allowing the top colourless aqueous phase to be removed cleanly using a P200 pipette- the transfer of a little of the organic phase by inaccurate pipetting should not be a problem as it will be removed by the second chloroform treatment. The aqueous phase was then transferred to a fresh microcentrifuge tube (1.5mL Alpha Laboratories) and re-extracted with an equal volume of chloroform (centrifuging at 13,000rpm for 15min at 4C). The aqueous phase was transferred to a fresh microcentrifuge tube, 0.8vol isopropanol added, mixed by inverting twice and incubated overnight at -20C to precipitate the nucleic acids. Nucleic acids may be precipitated for several hours at -70C or on dry ice, however overnight at -20C is recommended. It is not necessary to add additional salt to increase precipitation efficiency, and the use of coloured precipitation reagents is not recommended. The nucleic acid extractions were centrifuged at 13,000rpm for 20min at 4C. A white nucleic acid pellet should be visible from culture volumes of >50mL, the pellet may be invisible with lower numbers of bacilli. The supernatants were carefully removed (by pipetting) and discarded. The pellets were washed by adding 500µL cold 70% ethanol, centrifuging at 13,000rpm for 15min at 4C and removing the ethanol by pipetting. The tubes were respun briefly and any excess ethanol removed. The nucleic acid pellets were air dried at room temperature for 5-10min and then resuspended in RNase-free water. The preparations were then stored briefly on ice before continuing with the RNA clean up. 100µL RNase-free water was used to resuspend the nucleic acids extracted from every 100mL of mid-log phase culture volume and cleaned up using a single RNeasy column (i.e. 100mL culture divided into two ribolyser tubes, precipitated in two tubes, resuspended in 2x50µL and cleaned up on one RNeasy column). Incubation for 5-10min at room temperature with gently tapping should be sufficient to resuspend the nucleic acid. RNA preparations may be stored at -70C at this point, although it is recommended to continue immediately with RNA clean up to avoid freeze-thaw cycles. RNA Clean Up The nucleic acid samples were DNase I treated and purified using the RNeasy Mini Columns for RNA clean up (Qiagen), largely following manufacturers instructions. 350µL RLT buffer was added to each 100µL RNA sample and mixed thoroughly by pipetting (a final concentration of 10µl/mL B-mercaptoethanol was added to buffer RLT immediately before use). 250µL ethanol was added to the mixture, the solution mixed thoroughly by pipetting and applied immediately to the RNeasy Mini columns placed in 2mL collection tubes. The columns were centrifuged for 15s at 10,000rpm, and the flow-through and collection tubes discarded. The RNeasy mini columns were transferred to new 2mL tubes, and 350µL RWI buffer applied to the columns. The columns were centrifuged for 15s at 10,000rpm and the wash solution discarded. The samples were DNase I treated on the columns using the Qiagen RNase-free DNase kit. 80µL DNase I:buffer RDD mix was pipetted directly onto the column matrix (10µL DNase I plus 70µL RDD buffer which were combined before adding to the column), and the columns incubated at room temperature for 15min to remove contaminating DNA. A minimum incubation time of 15min is recommended (30min maximum). If RNA samples are to be used for applications that are particularly sensitive to genomic DNA such as quantitative RT-PCR, DNase I treatment may be repeated after washing with RW1 buffer. The columns were then washed by adding 350µL buffer RWI and centrifuging for 15s at 10,000rpm. 500µL of RPE buffer was applied to each column, the tubes centrifuged for 15s at 10,000rpm and the flow through discarded. A further 500µL buffer RPE was added to each column, before centrifuging for 2min at 10,000rpm. The columns were placed in new 2mL collection tubes and centrifuged for an additional 1min at 13,000 rpm to prevent carry-over of ethanol. The columns were transferred to new 1.5mL microcentrifuge tubes, 30µL RNase-free water was pipetted directly onto the column membrane and incubated at room temperature for 5min, before centrifuging for 1min at 10,000 rpm to elute the RNA. The eluate was re-applied to the column, incubated at room temperature for a further 5min and then centrifuged for 1min at 10,000rpm. The eluates from multiple RNA preparations from the same bacterial culture were pooled. The RNA was stored at -70C. RNA samples were quantified and the size distribution of product analysed using the Nanodrop Spectrophotometer and Agilent 2100 Bioanalyzer NanoChip systems. Elution volumes should be a minimum of 30µL, maximum 100µL. A second elution is recommended to increase RNA yield. Analyse RNA samples immediately before freezing or save two 2µL aliquots of each RNA preparation for Nanodrop and Bioanalyser analysis to avoid freeze thaw cycles. 500mL 5M GTC Solution 295.4g Guanidine thiocyanate (Promega V2791) 2.5g N-lauroyl-sarcosine (Sigma L5777) 12.5mL 1M sodium citrate (pH 7.0) 5mL Tween 80 (Sigma P8074) 3.5mL B-mercaptoethanol Add GTC powder to graduated 500mL flask. Add approximately 200mL dH2O, mix and leave in warm room overnight (reaction is endothermic, GTC dissolves slowly - shake occasionally). DO NOT MICROWAVE TO HEAT - generates cyanide gases. When GTC powder has dissolved add remaining constituents (except B-mercaptoethanol). Adjust volume to 500mL by adding dH2O. Store at room temperature. Add B-mercaptoethanol before use. Discard if GTC Solution develops a yellow colour.
100 ml 4 day log-phase M. tuberculosis cultures were treated with methionine sulfoximine for 4 h and 8 h alongside a carrier control (CC;100 μl H2O). Methionine Sulfoximine stock 0.2M in dH2O was filter sterilised and 100 ul added to 100 ml culture giving a final concentration of 200 μM. The dH20 control was also dilutd and filter sterilised.
Three 10ml cultures of M. tuberculosis H37Rv were grown at 37C in Middlebrook 7H9 broth (Difco) plus 10% (vol/vol) oleic acid-albumin-dextrose-catalase 2 (OADC) supplement (Becton Dickinson) and 0.05% (wt/vol) Tween 80. After 7 days the cultures were combined and OD taken. Four 100 ml volume 7H9 cultures were inoculated with 3 ml 7 day culture (passage 2-5, O.D approx 0.3), and incubated with stirring at 280 rpm using 40x8 mm stir bars for 4 days (ensuring screw top to 250 ml Duran bottle was loose enough to allow air exchange). O.D/CFU readings were taken from cultures (from day 4 to day 6) to ensure bacilli were in log-phase. Blood agar plates were set up on day 0 and day 4 to test for contamination.
DNA from 3 seperate amplifications were pooled.
Mycobacterium tuberculosis H37Rv genomic DNA (from Colorado State University) was amplified following manufacturers instructions using the Qiagen REPLI-g Mini Kit (150023). 25ng genomic DNA (in 2.5ul vol) was placed into a microcentrifuge tube, and 2.5ul Buffer D1 was added and mixed by vortexing. The samples were incubated at room temperature for 3min before 5ul Buffer N1 was added, vortexed and centrifuged briefly. 40ul of master mix containing reaction buffer, DNA polymerase and nuclease-free water was added, and the samples incubated for 16h at 30C. The DNA polymerase was inactivated by heating to 65C for 3min. The product from multiple reactions was combined, and the concentration was checked using the Nanodrop Spectrophotometer (found to be approx 0.3ug/ul). The amplified DNA was chloroform purified (with an equal volume of chloroform, vortexed, and centrifuged for 15min), and precipitated overnight with 0.8 vol isopropanol, 1/10 vol 3M sodium acetate. The samples were centrifuged, and the pellets washed with 70% ethanol. After air-drying briefly, the DNA was resuspended in 1xTE (left overnight and heated to 65C for 15min to aid resuspension). The concentration was measured using the Nanodrop Spectrophotometer, and diluted to 0.16ug/ul. The amplified genomic DNA was stored at 4C. 12ul (2ug aDNA) was added to each microarray hybridisation.
Standard BuG@S Affymetrix 428 scanning protocol. Parameters are read from the tif file during the upload process.