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冻融循环对肝癌新鲜冻存组织RNA完整性的影响
作者:张园1  侯彦深2  宋佳1  董翔1 
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
关键词:冻融 RNA提取时间 RNA完整性 
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:

[1] SHABIHKHANI M,LUVEY G M,WEI B,et al.The procurement,storage,and quality assurance of frozen blood and tissuebiospecimens in pathology,biorepository,and biobanksettings[J].Clin Biochem,2014,47(4-5):258-266.
[2] SCHROEDER A,MUELLER O,STOCKER S,et al.The RIN:an RNA integrity number for assigning integrity values to RNA measurements[J].BMC Mol Biol,2006,7:3.
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