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Microbiology 148 (2002), 2567-2572
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Microbiology (2002), 148, 2567-2572.
© 2002 Society for General Microbiology

Research Paper

Priming reverse transcription with oligo(dT) does not yield representative samples of Mycobacterium tuberculosis cDNA

David L. Lakey1,2,3, Yueru Zhang1, Adel M. Talaat4, Buka Samten1, Lucy E. DesJardin5, Kathleen D. Eisenach5, Stephen A. Johnston4 and Peter F. Barnes1,2,3

Center for Pulmonary and Infectious Disease Control1, and Departments of Microbiology and Immunology2 and Medicine3, University of Texas Health Center, Tyler, TX 75708, USA
Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA4
Department of Pathology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Health Care System, Little Rock, AR 72205, USA5

Author for correspondence: Peter Barnes. Tel: +1 903 877 7790. Fax: +1 903 877 5516. e-mail: peter.barnes{at}uthct.edu

Several recent publications have suggested that oligo(dT) can prime reverse transcription of several mycobacterial mRNAs. To determine if this is the case for most Mycobacterium tuberculosis mRNA species, reverse transcription reactions of M. tuberculosis RNA were primed with oligo(dT) or with other primers that did not target polyadenylylated sequences, and the resultant cDNA product was evaluated. Priming with oligo(dT) yielded more cDNA than priming with an arbitrary primer for only 1 of 12 unrelated M. tuberculosis genes, as measured by competitive PCR. Priming with oligo(dT) yielded cDNA for only 30% of the genes primed for by 37 M. tuberculosis genome-directed oligonucleotides, as assessed by hybridization of cDNA with an M. tuberculosis microarray. These data demonstrate that priming of reverse transcription of mycobacterial mRNA with oligo(dT) does not yield representative samples of cDNA.

Keywords: mycobacteria, gene expression, transcription

Abbreviations: gDNA, genomic DNA




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