Premium
Evaluation of quantitative polymerase chain reaction‐based approaches for determining gene copy and gene transcript numbers in environmental samples
Author(s) -
Smith Cindy J.,
Nedwell David B.,
Dong Liang F.,
Osborn A. Mark
Publication year - 2006
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2005.00963.x
Subject(s) - biology , serial dilution , microbiology and biotechnology , gene , polymerase chain reaction , real time polymerase chain reaction , complementary dna , nucleic acid , replicate , taq polymerase , gene expression , standard curve , 16s ribosomal rna , melting curve analysis , ribosomal rna , genetics , biochemistry , statistics , alternative medicine , mathematics , pathology , medicine , thermus aquaticus
Summary 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 Taq Man 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.