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J.D. McPherson

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    P1.03 - Poster Session 1 - Technology and Novel Development (ID 150)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.03-004 - Robust Global microRNA Expression Profiling Using Next-Generation Sequencing Technologies (ID 2063)

      09:30 - 16:30  |  Author(s): J.D. McPherson

      • Abstract

      MicroRNAs (miRNA) are endogenous, small regulatory nucleotides that negatively regulate gene expression post-transcriptionally. They are involved in a wide range of cellular functions, including growth, development, and apoptosis. Given their widespread roles in biological processes, changes in their expression are likely to be associated with the development and progression of diseases. Understanding their patterns of expression could provide new insights into complex biological processes and the possible clinical implications of miRNA dysfunction. As such, global miRNA expression profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used microarrays and quantitative real-time PCR (qRT-PCR). With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options.

      To compare the attributes of different profiling methodologies, five pairs of non-small cell lung cancer cell lines and their corresponding xenograft models were analysed using a microarray platform (Illumina Human microRNA Expression Profiling v2), NGS (Applied Biosystems SOLiD™ 3 Plus and 4 Systems and Illumina HiSeq2500), and the NanoString nCounter System (Human miRNA Expression Assay v1). The platforms were evaluated according to the following criteria: (i) inter-platform concordance, (ii) concordance with an independent validation method, qRT-PCR, and (iii) detection of differentially expressed miRNAs in a biologically relevant setting.

      Inter-platform correlations ranged from 0.62 – 0.80, while correlations with qRT-PCR, the current gold standard for validating expression profiling studies, was highly statistically significant for all platforms, with Spearman’s ρ ranging from 0.79 – 0.86. The accuracy in detecting differential expression was the highest for NGS (88%). Overall, sequencing technologies had the greatest detection sensitivity, along with the largest dynamic range of detection, and highest concordance with qRT-PCR. To assess the technical reproducibility of NGS, the same set of samples was profiled in duplicates. Using unsupervised hierarchical clustering, technical replicates for each biological sample clustered together, with Spearman’s ρ > 0.93 in all cases. miRNA analysis of formalin-fixed paraffin-embedded tissue (FFPE) was also evaluated. FFPE samples represent a rich source of archived specimen for retrospective studies of human disease. The feasibility of miRNA analysis with FFPE tissues would offer many opportunities to evaluate such large banks of archival materials. Three pairs of matched frozen and FFPE xenografts tumors were profiled using the Illumina HiSeq2000 platform. Hierarchical clustering showed similarity between expression profiles of paired frozen and FFPE samples (Spearman’s ρ > 0.88); whereas, samples of different biological origin were less correlated (Spearman’s ρ < 0.81).

      These results show the superior sensitivity, accuracy and robustness of NGS for global miRNA profiling in both frozen and FFPE tissue. Although microarrays and qRT-PCR have been used more extensively for expression profiling and are highly reproducible, they are limited to the detection of only known targets identified at the time of assay development and manufacturing. With the rapid increase in miRNAs being discovered and deposited in public databases, sequencing will offer a more comprehensive view of the miRNA transcriptome.