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Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions.

Cusick KD, Fitzgerald LA, Cockrell AL, Biffinger JC - PLoS ONE (2015)

Bottom Line: However, the molecular mechanisms associated with these key metabolic pathways remain unknown.Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB.Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.

View Article: PubMed Central - PubMed

Affiliation: National Research Council Associateship, US Naval Research Laboratory, 4555 Overlook Ave., SW, Washington DC, 20375, United States of America.

ABSTRACT
The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.

No MeSH data available.


Related in: MedlinePlus

Boxplot of CT values of candidate reference genes under all culture conditions.A line across the box depicts the median. The box indicates the 25th and 75th percentiles. Whiskers represent the 10th and 90th percentiles. Filled circles represent 5th and 95th percentiles.
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pone.0131015.g001: Boxplot of CT values of candidate reference genes under all culture conditions.A line across the box depicts the median. The box indicates the 25th and 75th percentiles. Whiskers represent the 10th and 90th percentiles. Filled circles represent 5th and 95th percentiles.

Mentions: The compliance of the RT-qPCR assays with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments [52]) guidelines is shown in the MIQE checklist (S2 Table). All assays were specific for their intended gene target, as indicated by a single peak in the melt curve analysis (S2 Fig) and possessed similar PCR efficiencies (Table 2). The average CT values, when assimilating the data from all 11 genes, ranged from ca. 16.8–22.3 under anaerobic/aerobic growth over time; 17.2–24.4 when comparing anaerobic growth using different terminal electron acceptors; and 16.5–21.6 under different heating methods (i.e. oven or microwave incubation), and 16.7–22.3 under all conditions combined (Fig 1 and Tables 3–6).


Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions.

Cusick KD, Fitzgerald LA, Cockrell AL, Biffinger JC - PLoS ONE (2015)

Boxplot of CT values of candidate reference genes under all culture conditions.A line across the box depicts the median. The box indicates the 25th and 75th percentiles. Whiskers represent the 10th and 90th percentiles. Filled circles represent 5th and 95th percentiles.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482720&req=5

pone.0131015.g001: Boxplot of CT values of candidate reference genes under all culture conditions.A line across the box depicts the median. The box indicates the 25th and 75th percentiles. Whiskers represent the 10th and 90th percentiles. Filled circles represent 5th and 95th percentiles.
Mentions: The compliance of the RT-qPCR assays with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments [52]) guidelines is shown in the MIQE checklist (S2 Table). All assays were specific for their intended gene target, as indicated by a single peak in the melt curve analysis (S2 Fig) and possessed similar PCR efficiencies (Table 2). The average CT values, when assimilating the data from all 11 genes, ranged from ca. 16.8–22.3 under anaerobic/aerobic growth over time; 17.2–24.4 when comparing anaerobic growth using different terminal electron acceptors; and 16.5–21.6 under different heating methods (i.e. oven or microwave incubation), and 16.7–22.3 under all conditions combined (Fig 1 and Tables 3–6).

Bottom Line: However, the molecular mechanisms associated with these key metabolic pathways remain unknown.Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB.Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.

View Article: PubMed Central - PubMed

Affiliation: National Research Council Associateship, US Naval Research Laboratory, 4555 Overlook Ave., SW, Washington DC, 20375, United States of America.

ABSTRACT
The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.

No MeSH data available.


Related in: MedlinePlus