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Publications (4)10.06 Total impact

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    ABSTRACT: MicroRNAs (miRNAs) are short (~22 nucleotides), non-coding RNA molecules that post-transcriptionally regulate gene expression. As the miRNA field is still in its relative infancy, there is currently a lack of consensus regarding optimal methodologies for miRNA quantification, data analysis and data standardization. To investigate miRNA measurement we selected a panel of both synthetic miRNA spikes and endogenous miRNAs to evaluate assay performance, copy number estimation, and relative quantification. We compared two different miRNA quantification methodologies and also assessed the impact of short RNA enrichment on the miRNA measurement. We found that both short RNA enrichment and quantification strategy used had a significant impact on miRNA measurement. Our findings illustrate that miRNA quantification can be influenced by the choice of methodology and this must be considered when interpreting miRNA analyses. Furthermore, we show that synthetic miRNA spikes can be used as effective experimental controls for the short RNA enrichment procedure.
    BioTechniques 03/2013; 54(3):155-64. · 2.40 Impact Factor
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    ABSTRACT: Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed.
    Methods 07/2012; · 3.64 Impact Factor
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    ABSTRACT: The application of toxicogenomics as a predictive tool for chemical risk assessment has been under evaluation by the toxicology community for more than a decade. However, it predominately remains a tool for investigative research rather than for regulatory risk assessment. In this study, we assessed whether the current generation of microarray technology in combination with an in vitro experimental design was capable of generating robust, reproducible data of sufficient quality to show promise as a tool for regulatory risk assessment. To this end, we designed a prospective collaborative study to determine the level of inter- and intra-laboratory reproducibility between three independent laboratories. All test centres (TCs) adopted the same protocols for all aspects of the toxicogenomic experiment including cell culture, chemical exposure, RNA extraction, microarray data generation and analysis. As a case study, the genotoxic carcinogen benzo[a]pyrene (B[a]P) and the human hepatoma cell line HepG2 were used to generate three comparable toxicogenomic data sets. High levels of technical reproducibility were demonstrated using a widely employed gene expression microarray platform. While differences at the global transcriptome level were observed between the TCs, a common subset of B[a]P responsive genes (n=400 gene probes) was identified at all TCs which included many genes previously reported in the literature as B[a]P responsive. These data show promise that the current generation of microarray technology, in combination with a standard in vitro experimental design, can produce robust data that can be generated reproducibly in independent laboratories. Future work will need to determine whether such reproducible in vitro model(s) can be predictive for a range of toxic chemicals with different mechanisms of action and thus be considered as part of future testing regimes for regulatory risk assessment.
    Toxicology 08/2011; 290(1):50-8. · 4.02 Impact Factor
  • Toxicology Letters - TOXICOL LETT. 01/2010; 196.