Author of

• 10923

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  P1.18 - Poster Session 1 - Pathology (ID 175)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Pathology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 10936

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    P1.18-013 - Extracting High quality RNA from FFPE samples for gene expression studies (ID 2092)

    09:30 - 09:30  |  Author(s): J. Weiss
    • Abstract

    Background
    The use of targeted therapies in the treatment of non-small cell lung cancer is still limited to a relatively small fraction of patients. Chemotherapy remains the mainstay of treatment for most patients today. So far, the best predictors for chemotherapeutic success are based on the expression of certain nucleotide metabolism genes or DNA damage response and repair related genes. However, the samples available for study most commonly comprise FFPE samples, which are characterised by a high degree of RNA fragmentation or degradation.

    Methods
    To address this problem, we have developed a protocol to reliably extract reasonable-quality RNA from FFPE samples. The protocol includes pathology review of the FFPE block, removal of a 2mm core, followed by RNA extraction. Next, the total RNA amount is quantified and a small proportion is accessed for fragmentation e.g. by TapeStation technology and/or a multiplex RT-PCR to determine the amount and size of amplifiable templates. We then assessed the extracted total RNA by various RNA based methodologies.

    Results
    To this end, we prepared core punches from 118 different lung adenocarcinomas and successfully extracted sufficient amounts of total RNA (> 50ng /ul in a 20ul elution) from 111 of the cores (average is 307ng/ul ranging from 53ng to 1.1ug/ul). Fragmentation assessment of 26 of these RNAs showed that all samples contained sufficient amounts of fragments with at least >100 nt. We first tested single gene expression by RT-qPCR. Of 26 samples tested, 24 samples showed robust amplification of a 161 bp fragment of the TBP housekeeping mRNA. We next assessed our RNA using gene expression analysis by NanoString®. We interrogated 150ng total RNA from 10 samples for the expression levels of 45 genes. Data analysis showed robust expression values and no quality control problems in all samples. Finally, we tested whether the RNA was of sufficient quality for next-generation RNA sequencing. We used 100 and 50 bp paired end sequencing on un-size-selected RNA, and 100 bp paired end sequencing after one round of size selection. On average, we obtained 23 million reads per sample, of which 70% mapped to reference sequences after either extensive read clipping or size selection.

    Conclusion
    In conclusion, our extraction protocol enables us to reliably extract total RNA from FFPE samples, which can be used for single-gene expression by RT-qPCR and gene expression of limited gene sets by NanoString® technology. However, the amount of samples and genes tested here were not sufficient to allow identification significant differences between samples, but shows the possibility to use the RNA extracted following our extraction protocol. RNAseq, however, poses a larger problem. The amount of mapped reads is significantly lower compared to high quality RNA from e.g. fresh frozen material or cell lines. The reason for these problems and possible solutions remain elusive. Overall, we present a simple and fast way to accurately extract RNA from FFPE material and show that after QC, single or small gene panels can successfully be assessed. However, large-scale sequencing efforts remain problematic and further optimization is needed.