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

doi:10.1186/1471-2199-10-63

BMC Molecular Biology
Volume 10

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Holmes DS
Quatrini R

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Research article

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

Pamela A Nieto¹ , Paulo C Covarrubias² , Eugenia Jedlicki¹ , David S Holmes²^,3 and Raquel Quatrini²

¹ICBM, Universidad de Chile, Santiago, Chile

²Center for Bioinformatics and Genome Biology, Fundación Ciencia para la Vida, MIFAB, Santiago, Chile

³Depto. de Ciencias Biologicas, Facultad de Ciencias de la Salud, Universidad Andres Bello, Santiago, Chile

author email corresponding author email

BMC Molecular Biology 2009, 10:63doi:10.1186/1471-2199-10-63


Published:	25 June 2009

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.