Journal Article

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

Redondo, Juliana, Moutasim, Karwan A, Kemp, Kevin C, Haynes, Harry R, Hares, Kelly M, Killick-Cole, Clare L, Wilkins, Alastair, Kurian, Kathreena M, Faulkner, Claire

2018

Overview

AimsHistopathological 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.MethodsTotal 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.ResultsReference 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.ConclusionsThis 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.

• Publisher: BMJ Publishing Group LTD
• Subject: Acids / Automation / Brain cancer / Brain Neoplasms - genetics / Brain Neoplasms - surgery / Breast cancer / Case-Control Studies / Epilepsy, Temporal Lobe - genetics / Epilepsy, Temporal Lobe - surgery / Fixatives - chemistry / Formaldehyde - chemistry / Gangrene / Gene expression / Gene Expression Profiling - standards / Glioblastoma - genetics / Glioblastoma - surgery / Humans / Morphology / Paraffin Embedding - standards / Predictive Value of Tests / Quality / Quality Control / Reproducibility of Results / RNA Stability / RNA, Neoplasm - genetics / Software / Time Factors / Tissue Fixation - methods / Tissue Fixation - standards