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

Aims: Histopathological tissue samples are being increasingly utilised as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control 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 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 vs 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 cycle threshold (Ct) 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 utilised in novel translational molecular biomarker studies.