- Methodology article
- Open Access
DNA-free RNA preparations from mycobacteria
© Stephan et al; licensee BioMed Central Ltd. 2004
- Received: 16 September 2004
- Accepted: 30 November 2004
- Published: 30 November 2004
To understand mycobacterial pathogenesis analysis of gene expression by quantification of RNA levels becomes increasingly important. However, current preparation methods yield mycobacterial RNA that is contaminated with chromosomal DNA.
After sonication of RNA samples from Mycobacterium smegmatis genomic DNA is efficiently removed by DNaseI in contrast to untreated samples.
This procedure eliminates one of the most prevalent error sources in quantification of RNA levels in mycobacteria.
- Mycobacterium Smegmatis
- Cesium Chloride
- Inactivation Step
- Guanidinium Isothiocyanate
- DNaseI Treatment
Mycobacterium tuberculosis is the leading cause of death from a single infectious disease with approximately 8.8 million new cases and two million deaths per year. To understand the pathogenesis of M. tuberculosis, analysis of gene expression by relative or absolute quantification of RNA levels using microarrays and RT-PCR (batch- and real-time) becomes increasingly important . Widely used methods to isolate bacterial RNA are acid-phenol extraction or guanidinium isothiocyanate extraction combined with cesium chloride purification or nucleic acid binding resins . However, the cell wall of mycobacteria is very stable and a very effective permeability barrier, and, therefore, rather refractory to lysis by chaotropic agents and detergents, hampering RNA isolation from these microorganisms . Since the average half-life of mycobacterial mRNA is in the range of a few minutes, mycobacteria have to be vigorously treated (e.g. bead-beating, freeze-thawing, nitrogen decompression) to quickly isolate RNA . This causes fragmentation of chromosomal DNA that contaminates RNA preparations, which is one of the most prevalent error sources in quantification of RNA levels in mycobacteria. Several methods have been suggested to circumvent this problem [5, 6]. Virtually all RNA isolation protocols use DNaseI, which does not completely remove large amounts of DNA. Our goal was to improve the efficiency of DNaseI digestion by solubilizing chromosomal DNA with sonication prior to DNaseI treatment. Mycobacterium smegmatis is especially refractory to lysis and therefore was chosen as a model organism.
This result shows that sonication improved DNA degradation by DNaseI most likely by rendering the chromosomal DNA more accessible to enzymatic action. This work describes a simple and efficient procedure to improve the quality of RNA preparations from M. smegmatis and will be of great value for RNA preparations from other microorganisms, including M. tuberculosis.
This work was supported by the Deutsche Forschungsgemeinschaft (NI 412, SFB 473). We thank Greer Kaufmann for editing the manuscript.
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