冻融循环对肝癌新鲜冻存组织RNA完整性的影响-东南大学学报医学版

冻融循环对肝癌新鲜冻存组织RNA完整性的影响

单位：1. 新疆医科大学附属肿瘤医院肿瘤防治研究所, 新疆维吾尔自治区乌鲁木齐 830011;
2. 新疆医科大学附属肿瘤医院麻醉科, 新疆维吾尔自治区乌鲁木齐 830011

分类号：R34

出版年·卷·期（页码）：2017·36·第一期（58-61）

摘要：

目的：评估新鲜冻存组织解冻后RNA降解动力学变化，探讨反复冻融对肝癌组织样本质量的影响。方法：将15例肝癌患者术后组织标本各分11份，其中1份用于组织形态学验证，另外10份于-80℃保存，取4份标本分别在冻融0、1、3、6次后提取RNA，剩余6份标本分别在室温下放置0min（T0）、5min（T5）、15min（T15）、30min（T30）、45min（T45）及1h（T60）后进行RNA提取，采用微芯片凝胶电泳技术分析RNA完整性。结果：切片中肿瘤组织面积均超过85%，坏死组织面积均小于15%，符合国际肿瘤基因组联盟制定的标准，且与术后病理学结果符合率达100%。不同冻融次数组织RNA完整性（RNA integrity number，RIN值）比较，差异有统计学意义（P<0.001），而且冻融次数越多，RIN值越低（冻融循环0、1、3、6次后RIN分别为8.27±0.42、6.38±0.61、5.08±0.61、4.32±0.62）。随解冻后RNA提取时间的延长，RNA完整性也不断降低（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），与T0时提取RNA比较，解冻后T30、T45、T60后提取RNA的RIN平均值显著下降，且差异有统计学意义（P<0.001）。结论：当研究对RNA质量要求比较高时，肝癌组织解冻不应超过3次，并且在30min内提取RNA，以保证RNA的完整性。

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.

参考文献：

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