title: Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements.
abstract: BACKGROUND
  Gene expression profiling is an important approach for detecting diagnostic and prognostic biomarkers, and predicting drug safety. The development of a wide range of technologies and platforms for measuring mRNA expression makes the evaluation and standardization of transcriptomic data problematic due to differences in protocols, data processing and analysis methods. Thus, universal RNA standards, such as those developed by the External RNA Controls Consortium (ERCC), are proposed to aid validation of research findings from diverse platforms such as microarrays and RT-qPCR, and play a role in quality control (QC) processes as transcriptomic profiling becomes more commonplace in the clinical setting.
  
  
  RESULTS
  Panels of ERCC RNA standards were constructed in order to test the utility of these reference materials (RMs) for performance characterization of two selected gene expression platforms, and for discrimination of biomarker profiles between groups. The linear range, limits of detection and reproducibility of microarray and RT-qPCR measurements were evaluated using panels of RNA standards. Transcripts of low abundance (≤ 10 copies/ng total RNA) showed more than double the technical variability compared to higher copy number transcripts on both platforms. Microarray profiling of two simulated 'normal' and 'disease' panels, each consisting of eight different RNA standards, yielded robust discrimination between the panels and between standards with varying fold change ratios, showing no systematic effects due to different labelling and hybridization runs. Also, comparison of microarray and RT-qPCR data for fold changes showed agreement for the two platforms.
  
  
  CONCLUSIONS
  ERCC RNA standards provide a generic means of evaluating different aspects of platform performance, and can provide information on the technical variation associated with quantification of biomarkers expressed at different levels of physiological abundance. Distinct panels of standards serve as an ideal quality control tool kit for determining the accuracy of fold change cut-off threshold and the impact of experimentally-derived noise on the discrimination of normal and disease profiles.
authors.affiliation: LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Alison S
authors.initials: AS
authors.lastname: Devonshire
authors.forename: Ramnath
authors.initials: R
authors.lastname: Elaswarapu
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
mesh_terms: D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis*; D004194:Disease / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D006801:Humans; D057230:Limit of Detection; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D012313:RNA