title: Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR.
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.
  
  
  CONCLUSIONS
  This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.
authors.affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore.
authors.forename: Kang
authors.initials: K
authors.lastname: Zhou
authors.forename: Lihan
authors.initials: L
authors.lastname: Zhou
authors.forename: Qing 'En
authors.initials: Q'
authors.lastname: Lim
authors.forename: Ruiyang
authors.initials: R
authors.lastname: Zou
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
authors.forename: Heng-Phon
authors.initials: HP
authors.lastname: Too
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000592:standards*; D005798:Genes, Bacterial; D053858:Metabolic Networks and Pathways; D016133:Polymerase Chain Reaction / Q000592:standards*; D040081:Protein Array Analysis / Q000592:standards; D011994:Recombinant Proteins / Q000235:genetics*; D012015:Reference Standards; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins