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

Smith, C. J. , Nedwell, D. B., Dong, L. F. and Osborn, A. M. (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. (doi: 10.1111/j.1462-2920.2005.00963.x)

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 r2 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.

• Item Type: Articles
• Additional Information: This work was conducted under a grant awarded to D.B.N.and A.M.O. from the UK Natural Environment ResearchCouncil (Ref. NER/A/S/2002/00962). This research also ben-efited from support from a grant awarded to AMO and Pro-fessor Kenneth Timmis from the Fifth Framework Programmeof the European Commission (Project EVK3-CT2002-00077,COMMODE)
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Smith, Professor Cindy
• Authors: Smith, C. J., Nedwell, D. B., Dong, L. F., and Osborn, A. M.
• College/School: College of Science and Engineering > School of Engineering > Infrastructure and Environment
• Journal Name: Environmental Microbiology
• Publisher: Society for Applied Microbiology and Blackwell Publishing Ltd
• ISSN: 1462-2912
• ISSN (Online): 1462-2920
• Published Online: 16 December 2005