Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

ABSTRACT: Background The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers. Results The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5ng total M.tuberculosis RNA with unamplified RNA from the same source. In addition a model M.tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons from an experimental system where RNA yield is restricted. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5ng total RNA (~ equivalent to 2E+05 bacilli). Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles. Conclusions Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-42

ORGANISM(S): Mycobacterium tuberculosis

SUBMITTER: Simon Waddell 

PROVIDER: E-BUGS-42 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress