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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

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 qua...

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Autores principales: Oldham, Athenia L., Duncan, Kathleen E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526484/
https://www.ncbi.nlm.nih.gov/pubmed/23284822
http://dx.doi.org/10.1371/journal.pone.0051931
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author Oldham, Athenia L.
Duncan, Kathleen E.
author_facet Oldham, Athenia L.
Duncan, Kathleen E.
author_sort Oldham, Athenia L.
collection PubMed
description 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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spelling pubmed-35264842013-01-02 Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model Oldham, Athenia L. Duncan, Kathleen E. PLoS One Research Article 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. Public Library of Science 2012-12-19 /pmc/articles/PMC3526484/ /pubmed/23284822 http://dx.doi.org/10.1371/journal.pone.0051931 Text en © 2012 Oldham, Duncan http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Oldham, Athenia L.
Duncan, Kathleen E.
Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model
title Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model
title_full Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model
title_fullStr Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model
title_full_unstemmed Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model
title_short Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model
title_sort similar gene estimates from circular and linear standards in quantitative pcr analyses using the prokaryotic 16s rrna gene as a model
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526484/
https://www.ncbi.nlm.nih.gov/pubmed/23284822
http://dx.doi.org/10.1371/journal.pone.0051931
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