Microarrays were scanned with a GenePix 4000B instrument (Axon Instruments, Inc.) with preliminary image analysis conducted using Imagene software (version 6.0, Biodiscovery).
Hybridization was carried out overnight (16-18 hr) at 45 degrees C. The hybridization buffer contains 5x SSC, 25% formamide, 0.15% SDS and 25 ug salmon sperm DNA.
In assays of binding to cytochalasin D-treated M?, 5 ug of unamplified RNA was used to generate labeled cDNA targets for microarray hybridization. CyDye fluors were conjugated to aa-cDNA using CyDye Post-Labeling Reactive Dye reagents (Amersham). For all other arrays, 10 ug of amino-allyl modified aRNA were labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen).
Using the MessageAmp-II Bacteria RNA Amplification system (Ambion), we amplified 100 ng total RNA to generate sufficient RNA from intracellular mycobacteria for microarray hybridization. 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 to generate multiple antisense RNA (aRNA) copies of each transcript. Amino-allyl UTP was incorporated into aRNA during the IVT reaction to permit fluorophore post-labeling.
Pelleted mycobacteria were lysed using 5 ug/ml lysozyme, hot Trizol, and physical disruption with silica beads in a BeadBeater. Total RNA was isolated by chloroform extraction followed by direct application to QIAGEN RNeasy column purification. Where indicated, M? were pretreated with 5 uM cytochalasin D or 100 nM concanamycin A for 20 min prior to infection to inhibit phagocytosis or phagosome acidification, respectively, and maintained throughout the infection period. Control mycobacteria not exposed to M? were treated with identical inhibitor concentrations.
M. turberculosis was grown in static cultures at 37 degrees C with starting and final optical density (OD) of 0.3 and 0.3-0.4 respectively. The culture media was Middlebrook 7H9 with oleic acid-albumin-glucose-catalase (OADC; Difco), Tween 80 and glycerol, pH 7, +/- 250 mM NaCl. For experimental (non-control) samples, the NaCl treatment lasted 4 hours.
Subsequent normalization, statistical analysis, and visualization of array data were performed with Genespring 7.3 (Agilent). Time-dependent changes in gene expression were analyzed using the EDGE methodology developed by Storey et al. (2005).