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Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays
  1. Harry R Haynes1,2,
  2. Clare L Killick-Cole3,
  3. Kelly M Hares4,
  4. Juliana Redondo4,
  5. Kevin C Kemp4,
  6. Karwan A Moutasim5,
  7. Claire Faulkner6,
  8. Alastair Wilkins4,
  9. Kathreena M Kurian1
  1. 1Brain Tumour Research Group, Institute of Clinical Neurosciences, University of Bristol, Bristol, UK
  2. 2Department of Cellular Pathology, North Bristol NHS Trust, Bristol, UK
  3. 3Functional Neurosurgery Research Group, Institute of Clinical Neurosciences, University of Bristol, Bristol, UK
  4. 4MS and Stem Cell Research Group, Institute of Clinical Neurosciences, University of Bristol, Bristol, UK
  5. 5Department of Cellular Pathology, University Hospital Southampton NHS Foundation Trust, Southampton, UK
  6. 6Bristol Genetics Laboratory, North Bristol NHS Trust, Bristol, UK
  1. Correspondence to Dr Harry R Haynes, Clinical Neuroscience, University of Bristol, Southmead Hospital, Bristol BS10 5NB, UK; harryrhaynes{at}doctors.org.uk

Abstract

Aims Histopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.

Methods Total RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, 18S and GAPDH.

Results Reference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.

Conclusions This study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

  • PCR
  • molecular pathology
  • brain tumours

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Footnotes

  • Handling editor Runjan Chetty.

  • Twitter Follow Harry R Haynes @hr_haynes.

  • Contributors Study conception and design: HRH, KMH, KCK, CF. Data collection, analysis and interpretation: HRH, CLKC, KMH, KCK, JR, CF. Manuscript production: HRH, CLKC, KAM, AW. Final approval of manuscript: HRH, AW and KMK.

  • Funding This work was supported by the Pathological Society and Jean Shanks Foundation Pathological Research Training Fellowship (HRH) and the Brain Tumour Bank South West (BRASH) at North Bristol NHS Trust UK. FFPE tissue samples were obtained from North Bristol NHS Trust as part of the UK Brain Archive Information Network (BRAIN UK), which is funded by the Medical Research Council and Brainstrust.

  • Competing interests None declared.

  • Ethics approval Control and disease tissue samples were provided under BrainUK ethical approval (14/008, 15/017).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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