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Validation of RNA integrity from low yield experiments with
Mycobacterium tuberculosis for downstream application
in real time PCR
Prathna Ramchandra and A. Willem Sturm*
Medical Microbiology Research Laboratory, Nelson R Mandela
School of Medicine, University of KwaZulu-Natal, South
Africa.
Corresponding author. E-mail:
sturm@ukzn.ac.za. Tel
+27 31 260 4391 / 4267.
Fax: +27 31 260 4527
Accepted 22 June, 2010 |
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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.
Key words: Mycobacteria, RNA extraction, RNA purification, quantitation. |
