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Open Access Highly Accessed Research article

Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

Kang Zhou1, Lihan Zhou12, Qing 'En Lim2, Ruiyang Zou1, Gregory Stephanopoulos13 and Heng-Phon Too12*

Author Affiliations

1 Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore

2 Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore

3 Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, The USA

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BMC Molecular Biology 2011, 12:18  doi:10.1186/1471-2199-12-18

Published: 23 April 2011

Abstract

Background

Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli.

Results

In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression.

Conclusion

This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.