Evaluation of a standardized protocol for the collection and storage of adrenal tumor samples – preparation for an international adrenal tumor bank

Tissue samples from adrenal tumors provide the basis for standard diagnostic procedures such as pathological examination. In addition, these tumor samples have been an invaluable source for the discovery of novel molecular pathways involved in that disease. Information on the molecular phenotype are based on DNA mutation analysis and epigenetic changes, RNA and protein expression pattern and sub-cellular localization as well as post transcriptional protein modification. However, storage and tissue handling of surgical adrenal tumor samples have not been optimized or standardized which might affect reproducibility and comparability between different laboratories. To examine different handling and storage procedures with regard to RNA, protein, and DNA quality and subsequent morphological examinations, we subdivided each surgical adrenal tumor sample into six pieces which were either snap frozen or treated with RNAlater (Ambion), after defined storage time at room temperature (up to 60min). DNA and protein recovery as well as integrity of DNA by means of pulse field electrophoresis and long range PCR (MC2-R locus) were not affected by snap freezing or the use of RNAlater after different storage intervals. Moreover, morphology of sectioned tissue samples was comparable between the groups, while overall staining intensity was decreased after RNAlater pre-treatment. In addition, western blotting did not reveal differences in the expression levels of 3ßHSD protein between the groups. However, levels of pERK as an example for a phosphorylated protein was significantly decreased by processing the tissue with RNAlater (snap vs. RNAlater after 15min storage, 100.0±17.6% vs. 57.7±6.4%, p=0.02). In summary, recovery and integrity of DNA and protein are not significantly affected by the investigated handling conditions while protein phosphorylation is dependent on pretreatment. Investigations of subtle differences in the RNA integrity and expression pattern are under way and will further define the optimal handling protocol of a scheduled European adrenal tumor tissue bank.