Priming reverse transcription with oligo(dT) does not yield representative samples of Mycobacterium tuberculosis cDNA.
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.
Lakey
David L
DL
Center for Pulmonary and Infectious Disease Control1, and Departments of Microbiology and Immunology2 and Medicine3, University of Texas Health Center, Tyler, TX 75708, USA.
Zhang
Yueru
Y
Center for Pulmonary and Infectious Disease Control1, and Departments of Microbiology and Immunology2 and Medicine3, University of Texas Health Center, Tyler, TX 75708, USA.
Talaat
Adel M
AM
Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA4.
Samten
Buka
B
Center for Pulmonary and Infectious Disease Control1, and Departments of Microbiology and Immunology2 and Medicine3, University of Texas Health Center, Tyler, TX 75708, USA.
DesJardin
Lucy E
LE
Department of Pathology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Health Care System, Little Rock, AR 72205, USA5.
Eisenach
Kathleen D
KD
Department of Pathology, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Health Care System, Little Rock, AR 72205, USA5.
Johnston
Stephen A
SA
Center for Biomedical Inventions, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA4.
Barnes
Peter F
PF
Center for Pulmonary and Infectious Disease Control1, and Departments of Microbiology and Immunology2 and Medicine3, University of Texas Health Center, Tyler, TX 75708, USA.
D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D012194:RNA-Directed DNA Polymerase