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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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Pipeline for the computational and experimental strategy used to identify suitable reference genes for normalization of expression.
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Figure 1: Pipeline for the computational and experimental strategy used to identify suitable reference genes for normalization of expression.

Mentions: The most useful reference genes for standards in gene expression studies should be stably expressed over a range of experimental conditions and should be of wide phylogenetic distribution. Taking these requirements into account, a combined computational and experimental approach was devised to identify reference genes in the Acidithiobacilli (Figure 1). The strategy involves the following steps:


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)

Pipeline for the computational and experimental strategy used to identify suitable reference genes for normalization of expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Pipeline for the computational and experimental strategy used to identify suitable reference genes for normalization of expression.
Mentions: The most useful reference genes for standards in gene expression studies should be stably expressed over a range of experimental conditions and should be of wide phylogenetic distribution. Taking these requirements into account, a combined computational and experimental approach was devised to identify reference genes in the Acidithiobacilli (Figure 1). The strategy involves the following steps:

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.

Show MeSH