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B.S. Paulsen



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    P2.17 - Poster Session 2 - Bronchoscopy, Endoscopy (ID 183)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pulmonology + Endoscopy/Pulmonary
    • Presentations: 1
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      P2.17-008 - A new procedure for sampling fine needle aspirations enables isolation of high quality mRNA (ID 2748)

      09:30 - 09:30  |  Author(s): B.S. Paulsen

      • Abstract

      Background
      Fine needle aspirates (FNA) are widely used in the diagnostics of non-small cell lung cancer (NSCLC). As relevant oncogenic drivers have emerged, the demand for molecular tests is increasing. To investigate mRNA expression fresh frozen tissue is required. In an everyday setting isolation of snap-frozen tissue is rarely a possibility and new procedures are needed to allow use of FNA material for mRNA expression studies.

      Methods
      Tumour samples from patients under suspicion of having lung cancer were isolated. Routine diagnostic procedures of FNA and/or transbronchial needle aspiration (TBNA) were performed and smears used for routine diagnostics were prepared. The remaining sample in the needle and syringe was mixed with 10 ml Cytolyt©. 9.5 ml was used for routine diagnostics, while 0.5 ml was mixed with 4.5 ml RNAprotect©. mRNA was isolated, transcribed into cDNA and quantified with quantitative reverse transcription quantitative PCR (RT-qPCR).

      Results
      Analysis of four reference genes in the first 50 samples consecutively collected showed that the most stable reference gene was β-actin. 211 tissue samples from 81 patients were then examined for β-actin mRNA expression. 92 % of the samples contained sufficient material for mRNA quantification containing mRNA corresponding to 100 HCC827 NSCLC cells or more. Furthermore 100 HCC827 NSCLC cells were sufficient to quantify the mRNA splice variant of BIM conferring erlotinib sensitivity.

      Conclusion
      We describe a novel procedure that optimizes the use of diagnostic FNA and TBNA and demonstrate that these samples contain enough material for isolation of high quality mRNA without influencing routine procedures.