Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4). MØ were infected (MOI ~20:1) with Mtb or BCG grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks. Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated. Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli. For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.
Growth protocol
The clinical isolate strain CDC1551 of M. tuberculosis (MTb) and the attenuated vaccine strain M. bovis BCG Pasteur (BCG) were routinely cultured in 7H9-OADC medium without shaking in 75 cm2 vented tissue culture flasks (T-75). Bone marrow-derived macrophages (MØ) were isolated from C57BL/6 mice and grown in DMEM supplemented with 10% FCS, 20% L cell-conditioned medium and antibiotics (penicillin and streptomycin). Macrophage media lacking antibiotics was added at least 24 hr before initiation of mycobacterial infections.
Extracted molecule
total RNA
Extraction protocol
Addition of a lysis buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate, and 0.1M -mercaptoethanol) selectively lysed MØ, halted RNA transcription/degradation while leaving mycobacteria intact as previously described (Butcher et al., 1998; Mangan et al., 2002). Samples were vortexed and passed five times through a 21 gauge needle to shear MØ and reduce viscosity. Intracellular mycobacteria were recovered by centrifugation at 3500 rpm for 30min. Pelleted BCG or MTb were digested with 5g/ml lysozyme before being lysed in 650C Trizol using a BeadBeater and 0.1mm silicon beads. Total RNA was isolated from Trizol lysates by chloroform extraction followed by addition of ethanol and direct application to Qiagen RNeasy column purification. Finally, residual DNA contamination was removed using Turbo DNAfree DNase (Ambion).
Label
Alexa 647
Label protocol
Using the MessageAmpTM-II Bacteria RNA Amplification system (Ambion), we amplified 100-250 ng total RNA to generate sufficient RNA from mycobacteria isolated from infected MØ for microarray hybrization. Briefly, total bacterial RNA was first polyadenylated by E. coli poly(A) polymerase. Using an oligo(dT) primer that incorporated a T7 promoter, poly(A) tailed RNA was reverse transcribed to yield double-stranded cDNA. The resulting cDNA served as the template for in vitro transcription (IVT) by T7 RNA polymerase which generates multiple antisense RNA (aRNA) copies of each transcript in the sample. Amino-allyl UTP was incorporated into aRNA during the IVT reaction to permit fluorophore post-labeling. According the manufacturer recommendation for consistent amplification results, a standardized RNA isolation protocol was used to generate all template RNA and master mixes of reaction components were prepared for each step. In addition, care was taken to use the same amount of starting template and identical incubation times and temperatures for all samples to be compared. amino-allyl modified aRNA were labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen) and purified using a MegaClear kit (Ambion).
Channel 2
Source name
Extracellular Control M. tuberculosis, 24hr in infection media, no macrophages
Prior to infections, bacteria were declumped by 10 passages through a 21 gauge needle and resuspended in infection buffer (DMEM with 5% fetal calf serum, 10mM HEPES, pH 7.4). MØ were infected (MOI ~20:1) with Mtb or BCG grown to mid-log phase (OD=0.6-0.8) in standing 75 cm2 vented tissue culture flasks. Infections were initiated by centrifugation (1000xg, 10 min) of bacteria onto confluent MØ monolayers followed by incubation at 370C for various times as indicated. Care was taken to treat control bacteria identically with the exception of the condition being tested to minimize detection of spurious changes in gene expression due to irrelevant stimuli. For example, for binding and invasion assays aliquots of the same bacterial samples were centrifuged and incubated in flasks without a MØ monolayer.
Growth protocol
The clinical isolate strain CDC1551 of M. tuberculosis (MTb) and the attenuated vaccine strain M. bovis BCG Pasteur (BCG) were routinely cultured in 7H9-OADC medium without shaking in 75 cm2 vented tissue culture flasks (T-75). Bone marrow-derived macrophages (MØ) were isolated from C57BL/6 mice and grown in DMEM supplemented with 10% FCS, 20% L cell-conditioned medium and antibiotics (penicillin and streptomycin). Macrophage media lacking antibiotics was added at least 24 hr before initiation of mycobacterial infections.
Extracted molecule
total RNA
Extraction protocol
Addition of a lysis buffer (4 M guanidine thiocyanate, 0.5% Na N-lauryl sarcosine, 25 mM sodium citrate, and 0.1M -mercaptoethanol) selectively lysed MØ, halted RNA transcription/degradation while leaving mycobacteria intact as previously described (Butcher et al., 1998; Mangan et al., 2002). Samples were vortexed and passed five times through a 21 gauge needle to shear MØ and reduce viscosity. Intracellular mycobacteria were recovered by centrifugation at 3500 rpm for 30min. Pelleted BCG or MTb were digested with 5g/ml lysozyme before being lysed in 650C Trizol using a BeadBeater and 0.1mm silicon beads. Total RNA was isolated from Trizol lysates by chloroform extraction followed by addition of ethanol and direct application to Qiagen RNeasy column purification. Finally, residual DNA contamination was removed using Turbo DNAfree DNase (Ambion).
Label
Alexa 555
Label protocol
Using the MessageAmpTM-II Bacteria RNA Amplification system (Ambion), we amplified 100-250 ng total RNA to generate sufficient RNA from mycobacteria isolated from infected MØ for microarray hybrization. Briefly, total bacterial RNA was first polyadenylated by E. coli poly(A) polymerase. Using an oligo(dT) primer that incorporated a T7 promoter, poly(A) tailed RNA was reverse transcribed to yield double-stranded cDNA. The resulting cDNA served as the template for in vitro transcription (IVT) by T7 RNA polymerase which generates multiple antisense RNA (aRNA) copies of each transcript in the sample. Amino-allyl UTP was incorporated into aRNA during the IVT reaction to permit fluorophore post-labeling. According the manufacturer recommendation for consistent amplification results, a standardized RNA isolation protocol was used to generate all template RNA and master mixes of reaction components were prepared for each step. In addition, care was taken to use the same amount of starting template and identical incubation times and temperatures for all samples to be compared. amino-allyl modified aRNA were labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen) and purified using a MegaClear kit (Ambion).
Hybridization protocol
5-10 ug of Cy-labeled Alexa-labeled aRNA from paired samples was dried using a Speedvac and resuspended in 50 l of hybridization buffer (5X SSC, 25% formamide, 0.1% SDS, and 25 g salmon sperm DNA). Samples were denatured at 95oC for 5 min and briefly cooled to 60oC before being applied to arrays under a glass LifterSlip (Erie Scientific). Slides were prehybridized for 1 hr in 25% formamide, 5X SSC, 0.1% SDS, 1% BSA and washed with H2O and isopropanol. Labeled targets were hybridized to microarrays in humidified slide chambers (Corning) at 45oC for 16-18 hr. Arrays were washed sequentially with buffer 1 (2X SSC, 0.1% SDS) pre-warmed to 45oC, buffer 2 (0.2X SSC, 0.1% SDS), buffer 3 (0.2X SSC), and buffer 4 (0.05X SSC). Slides were briefly dipped in de-ionized ultrafiltered water before being dried by centrifugation (700 X g, 10 min).
Scan protocol
Microarrays were scanned with a GenePix 4000B instrument (Axon Instruments, Inc.) with preliminary image analysis, spot intensity determination, background measurements, spot quality assessment and flagging conducted using Imagene software (version 6.0, Biodiscovery). PMT settings were adjusted to equalize the two channels based on equal intensity of positive control spots. PMT were also adjusted such that only a few spots were saturated to maximize detection of weak spots and maintain most intensities with linear range.
Description
nothing to add
Data processing
Poor quality spots with signal intensities less than three standard deviations above background were excluded from further analysis. Subsequent normalization, statistical analysis, and visualization of array data were performed with Genespring 7.3 (Agilent).
