Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples

Smith, Cindy J. and Nedwell, David B. and Dong, Liang F. and Osborn, A. Mark (2006) Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environmental Microbiology, 8 (5). pp. 804-815. ISSN 1462-2912

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Item Type:Article
Item Status:Live Archive

Abstract

Quantitative polymerase chain reaction (Q-PCR) amplification is widely applied for determining gene and transcript numbers within environmental samples. This research evaluated Q-PCR reproducibility via TaqMan assays quantifying 16S rRNA gene and transcript numbers in sediments, within and between replicate Q-PCR assays. Intra-assay variation in 16S rRNA gene numbers in replicate DNA samples was low (coefficients of variation; CV from 3.2 to 5.2). However, variability increased using replicated standard curves within separate Q-PCR assays (CV from 11.2 to 26), indicating absolute comparison of gene numbers between Q-PCR assays was less reliable. 16S rRNA transcript quantification was evaluated using standard curves of diluted RNA or cDNA (before, or following, reverse transcription). These standard curves were statistically different with cDNA-derived curves giving higher r 2 values and Q-PCR efficiencies. Template concentrations used in Q-PCR also affected 16S rRNA gene and transcript numbers. For DNA, 10 -3 dilutions yielded higher gene numbers than 10 -1 and 10 -2 dilutions. Conversely, RNA template dilution reduced numbers of transcripts detected. Finally, different nucleic acid isolation methods also resulted in gene and transcript number variability. This research demonstrates Q-PCR determination of absolute numbers of genes and transcripts using environmental nucleic acids should be treated cautiously. © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd.

Additional Information:Article first published online: 16 DEC 2005
Keywords:complementary DNA, RNA 16S, article, environmental monitoring, genetic transcription, genetics, methodology, reverse transcription polymerase chain reaction, sediment, soil, standard, DNA, Complementary, Geologic Sediments, Reverse Transcriptase Polymerase Chain Reaction, RNA, Ribosomal, 16S, Transcription, Genetic
Subjects:C Biological Sciences > C180 Ecology
C Biological Sciences > C500 Microbiology
Divisions:College of Science > School of Life Sciences
ID Code:8956
Deposited On:22 Apr 2013 12:18

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