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

• Published in: Microbiology (Society for General Microbiology) 2002-08, Vol.148 (8), p.2567-2572
• Main Authors: Lakey, David L, Zhang, Yueru, Talaat, Adel M, Samten, Buka, DesJardin, Lucy E, Eisenach, Kathleen D, Johnston, Stephen A, Barnes, Peter F
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; DNA Primers - genetics; DNA, Complementary - biosynthesis; DNA, Complementary - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Microbiology; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Mycology; Oligodeoxyribonucleotides - metabolism; Oligonucleotide Array Sequence Analysis; Oligonucleotides; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA-Directed DNA Polymerase - metabolism; Transcription, Genetic
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-148-8-2567

Center for Pulmonary and Infectious Disease Control 1 , and Departments of Microbiology and Immunology 2 and Medicine 3 , University of Texas Health Center, Tyler, TX 75708, USA Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 4 Department of Pathology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Health Care System, Little Rock, AR 72205, USA 5 Author for correspondence: Peter Barnes. Tel: [phone]. Fax: [phone]. 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