Validation of RNA integrity from low yield experiments with Mycobacterium tuberculosis for downstream application in real time PCR


Prathna Ramchandra and A. Willem Sturm

RNA extraction from mycobacterial cells is more challenging than from any other cell type. We used the Trizol Reagent® with silica beads to disrupt cells of Mycobacterium tuberculosis H37Rv. This method requires minimum reagents and handling and therefore aids in maintaining RNA integrity. The efficiency of cell disruption by this method was verified by microscopic analysis of the lysate. The yield and purity were determined using the Nanodrop-1000. A total volume of 50 µl containing 224.7 ng/µL of RNA was obtained from 3.1 x 108 cells, grown under sub-optimal conditions. RNA was visualized using the gene genius optical system. A 6 week old undisturbed culture of M. tuberculosis H37Rv, gave an expression ratio of fdxA/16s of -2.094. This extraction method is suitable for use of RNA in quantitative experiments, even if the cell numbers from which the RNA is extracted are low

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