Cryopreservation-Altered Expression of RNA and Protein Markers in Biological Specimens

The preservation of DNA, RNA, and protein markers in biological specimens is essential for initial diagnosis, subsequent verification, and comparison, as well as for archival retention of pathological materials in modern molecular diagnostics and precision medicine. Considerable attention has been paid to the methods of collection, handling, and preparation of specimens for initial testing, but insufficient attention to the long-term specimen preservation for later verification, comparison, and archival retention. In the present study, we have investigated the changes of expressions of RNAs and proteins in Hep-G2 cell specimens after cryopreservation at -80°C and in liquid nitrogen. Storage temperature and different cryoprotective agent (CPA) solutions not only affect cell viability but also more importantly the retention of various molecular markers after storage as detected by western blot and real-time fluorescent quantitative polymerase chain reaction. While the presence of CPAs increased the survival rates of cells after cryopreservation as expected, there was no consistent trend observed with regard to the RNA expression measurements. The data have significant implications with regard to the accuracy and interpretation of acquired data from specimens that have been cryopreserved without RNA and protein stabilization and point to the need for rethinking the assumptions, strategies, and criteria of optimizing biological specimen cryopreservation in molecular diagnostics.