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Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies.

Waddell, SJ; Laing, K; Senner, C; Butcher, PD (2008) Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies. BMC GENOMICS, 9 (94). ISSN 1471-2164 https://doi.org/10.1186/1471-2164-9-94
SGUL Authors: Butcher, Philip David Laing, Kenneth

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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 both linked to T7 polymerase in vitro run off transcription. 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 5 ng total M. tuberculosis RNA with unamplified RNA from the same source. In addition, as a direct measure of the effectiveness of bacterial amplification for identifying biologically relevant changes in gene expression, a model M. tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5 ng total RNA (~equivalent to 2 x 105 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 using both methods. CONCLUSION: 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.

Item Type: Article
Additional Information: © 2008 Waddell et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Aerobiosis, Base Sequence, DNA Primers, Gene Expression Profiling, Genes, Bacterial, Genomics, Mycobacterium tuberculosis, Nucleic Acid Amplification Techniques, Oligonucleotide Array Sequence Analysis, RNA, Bacterial, Reproducibility of Results, Sensitivity and Specificity, Science & Technology, Life Sciences & Biomedicine, Biotechnology & Applied Microbiology, Genetics & Heredity, COMPLETE GENOME SEQUENCE, GENE-EXPRESSION, BACTERIAL RNA, IN-VIVO, INFECTION, TRANSCRIPTION, MACROPHAGES, ENVIRONMENT, ADAPTATION, BIOLOGY
SGUL Research Institute / Research Centre: Academic Structure > Infection and Immunity Research Institute (INII)
Journal or Publication Title: BMC GENOMICS
ISSN: 1471-2164
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Dates:
DateEvent
25 February 2008Published
Web of Science ID: WOS:000254607300003
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URI: http://sgultest.da.ulcc.ac.uk/id/eprint/1802
Publisher's version: https://doi.org/10.1186/1471-2164-9-94

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