Optimization Of Rna Extraction In Mycobacterium Tuberculosis For Studying Intracellular Gene Expression

Abstract

Mycobacterium tuberculosis is the leading cause of death due to infectious disease after Human immunodeficiency virus. There has been an upsurge in the incidence of tuberculosis since 1980s. In order to reverse this trend, there is need to understand the biology of the organism. This can be brought about by studying gene expression at transcriptional level. The success of this hinges on RNA of good quality. In this paper, five methods (hot phenol, sonication with guanidinium thiocyanate (GTC) solution, beadbeating method with Trizol, FastPrep machine with Divolab as detergent and GTC solution, and FastPrep machine with Trizol) of extracting RNA from bacteria were compared to find
which of the method would be suitable for mycobacteria. The study found that physical method of lysing bacteria was necessary for extraction of RNA from mycobacteria. FastPrep machine gave the highest yield and also provided the speed necessary for optimum RNA extraction. FastPrep and Trizol as reagent for extraction of RNA was applied to macrophage infected with M. tuberculosis (H37Rv) after removing the macrophage RNA. We were able to demonstrate the expression of dnaK gene in both intracellular and broth grown bacilli. The expression of dnaK gene was found to be downregulated in macrophage compared to broth.

African Journal of Clinical and Experimental Microbiology Vol. 10 (2) 2009: pp. 64-79