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  MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)

  • Event: WCLC 2013
  • Type: Mini Oral Abstract Session
  • Track: Biology
  • Presentations: 1
  • Moderators:S. Lu, P. Waring
  • Coordinates: 10/28/2013, 10:30 - 12:00, Bayside 204 A+B, Level 2

  • 11067

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    MO01.04 - Comparison of Microarray and RNA Sequencing Platforms for Profiling MicroRNAs in Formalin-Fixed, Paraffin-Embedded Non-Small Cell Lung Cancer Specimens (ID 3145)

    10:40 - 10:45  |  Author(s): S.K. Patnaik
    • Abstract
    • Slides

    Background
    MicroRNAs are useful biomarkers for various disease states, and their preservation in formalin-fixed, paraffin-embedded (FFPE) tissue makes them particularly useful for clinicogenetic studies. Although global microRNA expression in FFPE samples is routinely measured with microarrays, the utility of RNA sequencing for such profiling has yet to be established. In this study, to appraise the suitability of RNA sequencing, microRNAs in RNA from lung cancer FFPE samples were quantified by both a microarray and a sequencing platform.

    Methods
    The affinity spin column–based Roche High Pure FFPE RNA kit was used to extract total RNA from 8 resected stage I lung adenocarcinoma FFPE tumor specimens (~3 mm[3]) with ≥50% tumor content. RNA was quantified by RiboGreen fluorometric and absorbance spectrometric analysis at 260 nm, and its quality was examined by electrophoresis on an RNA Pico chip in an Agilent Bioanalyzer 2100. MicroRNAs in 120 ng of RNA were profiled using the 8x60K Agilent Human miRNA Microarray (release 16.0) platform. MicroRNAs were also quantified by use of the Illumina HiSeq 2000 sequencing system (1x 50 bp reads), with multiplexed sequencing libraries prepared using 1 ug of RNA with the Illumina Truseq Small RNA Preparation Kit (version 2.0). Microarray data were processed using the AgiMicroRna Bioconductor package in R. Sequencing data were demultiplexed using CASAVA software and were mapped against mature human microRNAs in the miRBase database (version 16) using STAR aligner software. Absolute microRNA count values were then normalized among samples by use of the edgeR Bioconductor package.

    Results
    Results of RiboGreen fluorometric analysis suggested that an average of 16 ug (range, 6-35 ug; SD, 8 ug) of RNA was obtained from the FFPE specimens. Significant degradation of RNA was observed, as expected, with Bioanalyzer RNA integrity number values between 1.9 and 2.5. An average of 1.3 million sequencing reads (range, 9.1-16.9 million; SD, 3.5 million) were obtained, but only 1.4% (range, 0.4%-2.1%; SD, 1.4%) of them mapped to known microRNAs. Of the 1205 human microRNAs detectable with the microarray platform, 302 were identified as expressed in the 8-sample set, and 593 were identified as expressed in the sequencing platform. For the 177 microRNAs detected by both microarray and sequencing methods, the interplatform Spearman correlation coefficient was >0.5 for only 51 of them. Reverse-transcription PCR assays are being performed to identify the platform that yields the most accurate microRNA profile.

    Conclusion
    MicroRNA profiling by RNA sequencing and microarray techniques produced different results. The RNA sequencing method described here does not appear to be suitable for profiling microRNAs in RNA from FFPE samples. It is possible that depletion of ribosomal RNA fragments from FFPE RNA samples may improve the quality of data obtained from RNA sequencing.

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