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.