M.tb RNA extraction and amplification

Mycobacterial RNA was extracted from tuberculous sputa as previously described using the GTC/Trizol method [20]. Briefly, sputum was thawed and bacterial pellets recovered from GTC by centrifugation at 1800 g for 30 minutes. Bacterial pellets were resuspended in Trizol (Life Technologies), disrupted using a ribolyzer (MP Biomedicals) and the nucleic acid recovered in the aqueous phase after addition of chloroform. The RNA preparations were purified and DNase-treated using RNeasy columns (Qiagen). Mycobacterial RNA yield and quality were assayed using the Nano-Drop ND-1000 Spectrophotometer (NanoDrop Technologies) and Agilent 2100 Bioanalyser (Agilent Technologies). RNA samples were amplified from 100 ng total RNA using the MessageAmp II Bacteria system (Life Technologies) [16, 28]. All sputum samples were extracted and amplified together to minimize technical variation.