Analysis of RNA integrity of hepatocellular carcinoma cells with different cryopreservation times

Abstract

Objective To evaluate the quality control system for cryopreservation of hepatocellular carcinoma cells under-80℃as time relapses. Methods Hepatocellular carcinoma tissue was frozen within 24 hours after harvesting and stored for three months, six months and one year under ultra-low temperature respectively (n = 12 for each group) . Hepatocellular carcinoma and paracancerous tissue were identified by HE Staining. Promega automatic nucleic acid extraction instrument was used to extract RNA from HCC cells. RIN (RNA integrity number) value and 28S/18S ratio was evaluated by Agilent 2100 Bioanalyzer. Results The percentage of tumor cells were found in more than 80﹪of the samples by HE staining, and were considered as qualified samples. The average RIN values and 28S/18S ratios for HCC tissue stored for three months, six months and one year were > 7.0 and > 1.5 respectively. Unpairedttest showed that RIN value of cells stored for one year was decreased significantly compared with that freshly collected samples (7.442±0.674vs8.617±0.769,P= 0.001) or samples stored for three months (7.442±0.674vs 8.275±0.617,P= 0.005) and six months (7.442±0.674vs8.175±0.970,P = 0.043) , suggestingthe presence of RNA degradation. There was no significant difference between the other groups (P> 0.5) . Conclusion Morphology for tumor cells, RIN value and 28S/18S ratio can be used as indicators for monitoring the quality of liver cancer samples. Sample quality remained good when stored at ultra-low temperatures for three months or six months, although there was a certain degree of RNA degradation for samples that stored for one year. Key words: Liver neoplasms; Histiocytes; RNA; Cryopreservation