Objective:To evaluate the kinetics of RNA degradation and the effect of quality after thawing in fresh frozen liver cancer tissue.Methods:Fifteen specimens were from the patients of liver cancer.Each specimen was divided into 11 parts,1 of them was used for histomorphological verification,the others 10 were preserved in -80℃.Four parts of the specimen underwent 0 time,1 time,3 times,6 times repetitive freezing and thawing cycles before RNA extraction.The other 6 parts were thawed at room temperature for 0min(T0),5min(T5),30min(T30),45min(T45),1h(T60) before RNA extraction.RNA integrity was analyzed by microchip gel electrophoresis.Results:The tumor tissue area in detected specimens was over 85%,necrotic tissue area was less than 15%,which meet ICGC standard.The recombination rate of postoperative pathological diagnosis was 100%.The liver tissue RIN value was significant different after different freeze-thawing cycles,and the RIN value was decreased with increasing freeze-thawing times.The RINs were 8.27±0.42,6.38±0.61,5.08±0.61,4.32±0.62 at 0,1,3 and 6 times freeze-thawing cycles,respectively.The RNA integrity was decreased with increasing delayed RNA extraction time(T0:RIN=8.33±0.52;T5:RIN=7.95±0.48;T15:RIN=7.09±0.40;T30:RIN=5.88±0.39;T45:RIN=5.06±0.39;T60:RIN=4.40±0.30).The RIN value at T0 was significantly higher than those that at T30,T45 and T60 (P<0.001).Conclusion:According to the RIN results,we recommend that liver cancer tissue should not be thawed more than 3 times for studies requiring RNA of high quality and within 30 minutes for RNA extraction. |