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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

Representative figure of data used to obtain transcript-to-gene ratios for gyrB, polA2, and rpoC.Samples were collected at multiple time points from anaerobic cultures supplemented with glucose as the carbon source and nitrate as the terminal electron acceptor. Transcript and gene copies per mL of sample were calculated via absolute quantification using external calibration curves. Error bars represent the standard deviation derived from triplicate qPCRs from three biological replicates.
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pone.0131015.g002: Representative figure of data used to obtain transcript-to-gene ratios for gyrB, polA2, and rpoC.Samples were collected at multiple time points from anaerobic cultures supplemented with glucose as the carbon source and nitrate as the terminal electron acceptor. Transcript and gene copies per mL of sample were calculated via absolute quantification using external calibration curves. Error bars represent the standard deviation derived from triplicate qPCRs from three biological replicates.

Mentions: External, PCR-amplicon-based standards were created and used to calculate both transcript and gene copy numbers per mL of sample in order to determine the ratio of transcript copies to gene copies under the various culture conditions (representative image in Fig 2, plots of transcript and gene copy number for all genes under all conditions S5–S13 Figs). This transcript: gene copy number ratio indicated whether CT fluctuations were due to changes in transcript abundance, indicative of changes in gene expression, or changes in gene copy number, such as would occur during mono/polyploidy transitions. As described in a previous study, the Pearson Product Correlation test was applied to the ratio and corresponding CT of each time point for each gene to test the hypothesis that a negative correlation exists between ratio and CT [36]. With the exception of gyrB across small-volume aerobic/anaerobic conditions (Table 7, “gyrB Growth”), statistically significant negative correlations were found to exist between the transcript: gene ratio and CT values among the three genes under all conditions (Table 7). These correlations provide strong evidence that CT changes are an accurate depiction of transcript copy number, and not gene copy number, fluctuations. Plotting both the transcript: gene copy number ratio and CT values over time illustrates that in general, CT changes reflect fluxes in transcript copy number (Figs 3–5). For example, when grown aerobically in an oven, the gyrB CT and ratio values showed an inverse trend over time (Fig 3E). If this were due to changes in gene (DNA) copy number, the ratio pattern would mirror that of the CTs. However, an increase in ratio is strongly indicative of an increase in transcript abundance. These data demonstrate that even with the potential for polyploidy, relative quantification is applicable for measuring gene expression changes in extremophiles.


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)

Representative figure of data used to obtain transcript-to-gene ratios for gyrB, polA2, and rpoC.Samples were collected at multiple time points from anaerobic cultures supplemented with glucose as the carbon source and nitrate as the terminal electron acceptor. Transcript and gene copies per mL of sample were calculated via absolute quantification using external calibration curves. Error bars represent the standard deviation derived from triplicate qPCRs from three biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131015.g002: Representative figure of data used to obtain transcript-to-gene ratios for gyrB, polA2, and rpoC.Samples were collected at multiple time points from anaerobic cultures supplemented with glucose as the carbon source and nitrate as the terminal electron acceptor. Transcript and gene copies per mL of sample were calculated via absolute quantification using external calibration curves. Error bars represent the standard deviation derived from triplicate qPCRs from three biological replicates.
Mentions: External, PCR-amplicon-based standards were created and used to calculate both transcript and gene copy numbers per mL of sample in order to determine the ratio of transcript copies to gene copies under the various culture conditions (representative image in Fig 2, plots of transcript and gene copy number for all genes under all conditions S5–S13 Figs). This transcript: gene copy number ratio indicated whether CT fluctuations were due to changes in transcript abundance, indicative of changes in gene expression, or changes in gene copy number, such as would occur during mono/polyploidy transitions. As described in a previous study, the Pearson Product Correlation test was applied to the ratio and corresponding CT of each time point for each gene to test the hypothesis that a negative correlation exists between ratio and CT [36]. With the exception of gyrB across small-volume aerobic/anaerobic conditions (Table 7, “gyrB Growth”), statistically significant negative correlations were found to exist between the transcript: gene ratio and CT values among the three genes under all conditions (Table 7). These correlations provide strong evidence that CT changes are an accurate depiction of transcript copy number, and not gene copy number, fluctuations. Plotting both the transcript: gene copy number ratio and CT values over time illustrates that in general, CT changes reflect fluxes in transcript copy number (Figs 3–5). For example, when grown aerobically in an oven, the gyrB CT and ratio values showed an inverse trend over time (Fig 3E). If this were due to changes in gene (DNA) copy number, the ratio pattern would mirror that of the CTs. However, an increase in ratio is strongly indicative of an increase in transcript abundance. These data demonstrate that even with the potential for polyploidy, relative quantification is applicable for measuring gene expression changes in extremophiles.

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