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Similar gene estimates from circular and linear standards in quantitative PCR analyses using the prokaryotic 16S rRNA gene as a model.

Oldham AL, Duncan KE - PLoS ONE (2012)

Bottom Line: Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples.Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively.The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.

View Article: PubMed Central - PubMed

Affiliation: The Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA.

ABSTRACT
Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples. A recent publication showed that circular plasmid DNA standards grossly overestimated numbers of a target gene by as much as 8-fold in a eukaryotic system using quantitative PCR (qPCR) analysis. Overestimation of microbial numbers is a serious concern in industrial settings where qPCR estimates form the basis for quality control or mitigation decisions. Unlike eukaryotes, bacteria and archaea most commonly have circular genomes and plasmids and therefore may not be subject to the same levels of overestimation. Therefore, the feasibility of using circular DNA plasmids as standards for 16S rRNA gene estimates was assayed using these two prokaryotic systems, with the practical advantage being rapid standard preparation for ongoing qPCR analyses. Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively. Estimates of 16S rRNA gene copies were made based on circular and linearized DNA conformations using two genomes from each domain: Desulfovibrio vulgaris, Pseudomonas aeruginosa, Archaeoglobus fulgidus, and Methanocaldocococcus jannaschii. The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.

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Comparison of expected and estimated 16S rRNA gene copies in bacterial DNA samples.Expected bacterial 16S rRNA gene copies were calculated based on four and five 16S copies per genome for (a) P. aeruginosa and (b) D. vulgaris, respectively. Black bars = predicted 16S copies. White bars = estimated 16S copies based on supercoiled plasmid standard. Grey bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon-based standard. Data are the average (n = 3) and error bars are ±1 standard deviation among replicates.
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pone-0051931-g003: Comparison of expected and estimated 16S rRNA gene copies in bacterial DNA samples.Expected bacterial 16S rRNA gene copies were calculated based on four and five 16S copies per genome for (a) P. aeruginosa and (b) D. vulgaris, respectively. Black bars = predicted 16S copies. White bars = estimated 16S copies based on supercoiled plasmid standard. Grey bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon-based standard. Data are the average (n = 3) and error bars are ±1 standard deviation among replicates.

Mentions: The estimated number of bacterial 16S rRNA gene copies, based on the four standard curves, was compared to predicted 16S rRNA gene copy numbers (Figure 3 and Table 4). For both bacterial genomes, gene estimates derived from nicked circles and linearized plasmids were indistinguishable from one another. For both archaeal genomes, estimates derived from both linear and circular standard curves approached 1 (Figure 4 and Table 4). Note that the A. fulgidus 16S rRNA gene sequence was used as the standard for the archaeal qPCR reactions and was expected to be a precise match. Interestingly, both circular plasmids provided the best estimates for the archaeal 16S rRNA gene. Taken together, these results demonstrate than no single standard conformation performed the best in all instances. Importantly, estimates using the supercoiled standard never approached the 8-fold overestimates noted for eukaryotic systems.


Similar gene estimates from circular and linear standards in quantitative PCR analyses using the prokaryotic 16S rRNA gene as a model.

Oldham AL, Duncan KE - PLoS ONE (2012)

Comparison of expected and estimated 16S rRNA gene copies in bacterial DNA samples.Expected bacterial 16S rRNA gene copies were calculated based on four and five 16S copies per genome for (a) P. aeruginosa and (b) D. vulgaris, respectively. Black bars = predicted 16S copies. White bars = estimated 16S copies based on supercoiled plasmid standard. Grey bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon-based standard. Data are the average (n = 3) and error bars are ±1 standard deviation among replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526484&req=5

pone-0051931-g003: Comparison of expected and estimated 16S rRNA gene copies in bacterial DNA samples.Expected bacterial 16S rRNA gene copies were calculated based on four and five 16S copies per genome for (a) P. aeruginosa and (b) D. vulgaris, respectively. Black bars = predicted 16S copies. White bars = estimated 16S copies based on supercoiled plasmid standard. Grey bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard. Black and gray striped bars = estimated 16S copies based on amplicon-based standard. Data are the average (n = 3) and error bars are ±1 standard deviation among replicates.
Mentions: The estimated number of bacterial 16S rRNA gene copies, based on the four standard curves, was compared to predicted 16S rRNA gene copy numbers (Figure 3 and Table 4). For both bacterial genomes, gene estimates derived from nicked circles and linearized plasmids were indistinguishable from one another. For both archaeal genomes, estimates derived from both linear and circular standard curves approached 1 (Figure 4 and Table 4). Note that the A. fulgidus 16S rRNA gene sequence was used as the standard for the archaeal qPCR reactions and was expected to be a precise match. Interestingly, both circular plasmids provided the best estimates for the archaeal 16S rRNA gene. Taken together, these results demonstrate than no single standard conformation performed the best in all instances. Importantly, estimates using the supercoiled standard never approached the 8-fold overestimates noted for eukaryotic systems.

Bottom Line: Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples.Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively.The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.

View Article: PubMed Central - PubMed

Affiliation: The Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA.

ABSTRACT
Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples. A recent publication showed that circular plasmid DNA standards grossly overestimated numbers of a target gene by as much as 8-fold in a eukaryotic system using quantitative PCR (qPCR) analysis. Overestimation of microbial numbers is a serious concern in industrial settings where qPCR estimates form the basis for quality control or mitigation decisions. Unlike eukaryotes, bacteria and archaea most commonly have circular genomes and plasmids and therefore may not be subject to the same levels of overestimation. Therefore, the feasibility of using circular DNA plasmids as standards for 16S rRNA gene estimates was assayed using these two prokaryotic systems, with the practical advantage being rapid standard preparation for ongoing qPCR analyses. Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively. Estimates of 16S rRNA gene copies were made based on circular and linearized DNA conformations using two genomes from each domain: Desulfovibrio vulgaris, Pseudomonas aeruginosa, Archaeoglobus fulgidus, and Methanocaldocococcus jannaschii. The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.

Show MeSH
Related in: MedlinePlus