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Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans.

Nieto PA, Covarrubias PC, Jedlicki E, Holmes DS, Quatrini R - BMC Mol. Biol. (2009)

Bottom Line: Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated.This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli.The information could also be of value for future transcriptomic experiments in other bacterial systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: ICBM, Universidad de Chile, Santiago, Chile. pamelanietop@gmail.com

ABSTRACT

Background: Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data.

Results: Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans.

Conclusion: This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems.

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Relative expression levels of alaS, recA and rrs and the nine candidate reference genes in all possible pairwise combinations (A/B) of five experimental conditions analyzed. Expression levels of the reference genes in each experimental condition (A) was compared to their respective expression levels in all other conditions evaluated (B). Boxes represent the average ratios (A/B) in all possible pairwise combinations of experimental conditions analyzed and bars represent the standard deviations of the average ratios A/B. White boxes correspond to reference genes selected after applying the strategy outlined in Figure 1. Black boxes correspond to reference genes previously used in A. ferrooxidans research [28,30,45].
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Figure 2: Relative expression levels of alaS, recA and rrs and the nine candidate reference genes in all possible pairwise combinations (A/B) of five experimental conditions analyzed. Expression levels of the reference genes in each experimental condition (A) was compared to their respective expression levels in all other conditions evaluated (B). Boxes represent the average ratios (A/B) in all possible pairwise combinations of experimental conditions analyzed and bars represent the standard deviations of the average ratios A/B. White boxes correspond to reference genes selected after applying the strategy outlined in Figure 1. Black boxes correspond to reference genes previously used in A. ferrooxidans research [28,30,45].

Mentions: The expression of recA, alaS and rrs was analyzed under the same experimental conditions and their expression values were ranked with respect to the new reference genes derived above using geNorm [11] and NormFinder [7] (Table 2). The variability in expression of rrs and alaS in five different experimental setups shows them to perform well although slightly poorer than rpoC, era and map (Figure 2). Both geNorm and NormFinder identified rrs and alaS among the six more stable genes. On the contrary, recA ranks further down, indicating less stable expression, independently of the ranking method used (Table 2). In addition, the expression of recA varies more than two fold, and together with coaE and trpS exhibits the least suitable expression profile of the genes assessed in this study (Figure 2).


Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans.

Nieto PA, Covarrubias PC, Jedlicki E, Holmes DS, Quatrini R - BMC Mol. Biol. (2009)

Relative expression levels of alaS, recA and rrs and the nine candidate reference genes in all possible pairwise combinations (A/B) of five experimental conditions analyzed. Expression levels of the reference genes in each experimental condition (A) was compared to their respective expression levels in all other conditions evaluated (B). Boxes represent the average ratios (A/B) in all possible pairwise combinations of experimental conditions analyzed and bars represent the standard deviations of the average ratios A/B. White boxes correspond to reference genes selected after applying the strategy outlined in Figure 1. Black boxes correspond to reference genes previously used in A. ferrooxidans research [28,30,45].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713239&req=5

Figure 2: Relative expression levels of alaS, recA and rrs and the nine candidate reference genes in all possible pairwise combinations (A/B) of five experimental conditions analyzed. Expression levels of the reference genes in each experimental condition (A) was compared to their respective expression levels in all other conditions evaluated (B). Boxes represent the average ratios (A/B) in all possible pairwise combinations of experimental conditions analyzed and bars represent the standard deviations of the average ratios A/B. White boxes correspond to reference genes selected after applying the strategy outlined in Figure 1. Black boxes correspond to reference genes previously used in A. ferrooxidans research [28,30,45].
Mentions: The expression of recA, alaS and rrs was analyzed under the same experimental conditions and their expression values were ranked with respect to the new reference genes derived above using geNorm [11] and NormFinder [7] (Table 2). The variability in expression of rrs and alaS in five different experimental setups shows them to perform well although slightly poorer than rpoC, era and map (Figure 2). Both geNorm and NormFinder identified rrs and alaS among the six more stable genes. On the contrary, recA ranks further down, indicating less stable expression, independently of the ranking method used (Table 2). In addition, the expression of recA varies more than two fold, and together with coaE and trpS exhibits the least suitable expression profile of the genes assessed in this study (Figure 2).

Bottom Line: Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated.This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli.The information could also be of value for future transcriptomic experiments in other bacterial systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: ICBM, Universidad de Chile, Santiago, Chile. pamelanietop@gmail.com

ABSTRACT

Background: Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data.

Results: Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans.

Conclusion: This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems.

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