张佳年①②, 计骏②, 刘炳亚②, 朱正纲②, 傅国辉①, 于颖彦②. 深低温冻存胃癌样本的核酸与蛋白质质量控制研究*[J]. 中国肿瘤临床, 2016, 43(1): 27-34. DOI: 10.3969/j.issn.1000-8179.2016.01.077
引用本文: 张佳年①②, 计骏②, 刘炳亚②, 朱正纲②, 傅国辉①, 于颖彦②. 深低温冻存胃癌样本的核酸与蛋白质质量控制研究*[J]. 中国肿瘤临床, 2016, 43(1): 27-34. DOI: 10.3969/j.issn.1000-8179.2016.01.077
Jianian ZHANG1, 2, Jun JI2, Bingya LIU2, Zhenggang ZHU2, Guohui FU1. Quality control of nucleic acids and protein of freeze-preserving gastric cancer samples[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2016, 43(1): 27-34. DOI: 10.3969/j.issn.1000-8179.2016.01.077
Citation: Jianian ZHANG1, 2, Jun JI2, Bingya LIU2, Zhenggang ZHU2, Guohui FU1. Quality control of nucleic acids and protein of freeze-preserving gastric cancer samples[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2016, 43(1): 27-34. DOI: 10.3969/j.issn.1000-8179.2016.01.077

深低温冻存胃癌样本的核酸与蛋白质质量控制研究*

Quality control of nucleic acids and protein of freeze-preserving gastric cancer samples

  • 摘要: 目的:探索上海市瑞金医院低温冻存0~ 10年胃癌样本的核酸与蛋白质质量。方法:随机抽取2003年至2014年间库存手术切除胃癌标本24对(共48份)。 1% 琼脂糖凝胶电泳检测DNA 与RNA 纯度及完整性,同时增加了RIN 值评估法检测RNA 完整性;BCA 法检测样本蛋白质浓度,考马斯亮蓝法测定蛋白质分子完整性。结果:将组织标本按冻存年限分成四组(< 2 年、3~5 年、6~8 年和> 9 年),各组之间DNA 完整性差异无统计学意义(P > 0.05),正常胃组织的DNA 降解程度比胃癌组织高(P = 0.023);四组之间的RIN 值检测显示,冻存6 年以上组的RIN 值有明显下降(P = 0.018);各组间的蛋白质浓度无显著性差异,考马斯亮蓝法测定蛋白质分子条带完整性存在显著性差异。冻存> 9 年组仅出现少量低分子量条带(平均 36.5 kD),冻存 6~8 年组尚有中等分子量(平均 65.63kD)条带,冻存3~5 年组有高分子量(平均127.5 kD)条带,冻存短于2 年组仍可见大分子量(平均160 kD)条带。结论:长期低温冻存对DNA 影响最小,超过5 年组RNA 和大分子量蛋白质降解较多,但中小分子量蛋白质保存完好。

     

    Abstract: Objective:To explore the quality of inventory samples of a biobank stored in a deep freezer from 0 to over 10years in Shanghai Ruijin Hospital.Methods:We extracted24pairs of stocked gastric cancer samples between 2003and 2014. We used 1% aga-rose gel electrophoresis to analyze DNA and RNA purity and integrity while adding the RNA integrity number (RIN) for precise analysis. Bicinchonininc acid (BCA) assay was used for protein concentration evaluation. Coomassie brilliant blue method was used for protein integrity assay. Results: The samples were divided into four groups according to cryopreservation period (<2 years, 3- 5 years, 6- 8 years, and > 9 years). No significant difference in DNA integrity was found between the groups (P>0. 05); however, DNA degradation in normal gastric mucosa was faster than that in gastric cancer tissue (P=0. 023 ). The RIN significantly declined when the storage period was 6 years or longer ( P=0. 018 ). No significant difference in protein concentration was observed between different groups. Using Coo -massie brilliant blue method, we found significant differences in preserved proteins with different molecular weights. Proteins with varying molecular weights were detected in the groups with the following cryopreservation periods: > 9 years, a small number of low-molecular-weight (average 36. 5 KD) proteins;6- 8 years, medium-molecular-weight (average65. 63KD) proteins;3- 5 years, high-molecu -lar-weight (average 127 . 5 KD) proteins; <2 years, high-molecular-weight (average 160 KD) proteins. Conclusion: Cryopreservation does not exert an obvious effect on DNA. If the cryopreservation period is more than 5 years, serious degradation of RNA should occur; like-wise, degradation of proteins with higher molecular weight should occur.

     

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