Effects of processing, storage time, and tissue volume in the retrieval and quality of RNA and the expression level of RNU6

Background: Molecular analyses of tumor RNA expression have become widely used both for research and clinical purposes. Tumoral tissue preservation is a critical step to ensure accuracy of molecular-based diagnostics, for which, formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source of clinical samples. MicroRNAs are ideal biomarkers in FFPE-tissues, in whose expression evaluation RNU6 is one of the genes used as a normalizer. Our aim was to determine, in FFPE tissue samples, the effects of length of storage and corresponding volume of each studied sample, on the RNA retrieval, quality and concentration, as well as their correlation to the expression level of RNU6. Methods: Fifty tissue blocks with a mean length of tissue storage of 30 months (SD=±12.07, 95% CI= 27.4-34.3). were included. Total RNA was isolated, absorbance and concentrations were determined and correlated with length of storage and volume of tissue. RT-qPCR for RNU6 was performed and their Ct results were correlated to the same parameters. Results: There was a direct correlation between the concentration and quality of the obtained RNA, and an inverse correlation between the tissue storage time and the RNA quality. The volume of tissue studied was not correlated with the RNA quality or concentration. The RNA quality and the length of tissue storage directly correlated to the RNU6 expression level, while RNA concentration and the volume of tissue studied did not affect it. Conclusions: There is an association between longer FFPE tissue storage time with lower RNA quality and lower RNU6 expression level.