A robust and efficient method for the isolation of DNA-free, pure and intact RNA from Mycobacterium tuberculosis

• A method for RNA preparation from mycobacteria with consistent yields is proposed.
• The method yields 1 μg to 3 μg total RNA per 107 cells of mycobacteria.
• It involves enhanced lysis of mycobacteria with efficient removal of genomic DNA.
• RNA prepared by this method gave reproducible results in real time PCR.
• The RNA obtained is suitable for high-throughput sequencing after rRNA depletion.

We describe a robust method for the isolation of pure, intact RNA suitable for transcriptome studies from mycobacteria with consistent yields of 1 μg to 3 μg total RNA per 107 cells. The method reduces the use of hazardous chemicals and incorporates protocols for efficient removal of gDNA and rRNA.