张佳年　计骏　刘炳亚　朱正纲　傅国辉　于颖彦



深低温冻存胃癌样本的核酸与蛋白质质量控制研究*

张佳年①②计骏②刘炳亚②朱正纲②傅国辉①于颖彦②

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65.63 kD）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。

关键词生物样本胃癌低温保存核酸蛋白质质量控制

*本文课题受国家高技术研究发展计划“863”项目（编号：2012AA02A504，2012AA02A203）、国家自然科学基金项目（编号：81172329，81372644）、上海交通大学医学院转化医学创新基金（编号：15ZH1002，15ZH4001）和“985”生物样本库建设基金资助

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

Jianian ZHANG1,2, Jun JI2, Bingya LIU2, Zhenggang ZHU2, Guohui FU1, Yingyan YU2
Correspondence to: Yingyan YU, E-mail: ruijinhospitalyyy@163.com; Guohui FU, E-mail: fuguhu@263.net
1Department of Pathology, Basic Medical College, Shanghai Jiao Tong University;2School of Medicine and Shanghai Ruijin Hospital, Shanghai Institute of Digestive Surgery, the Key Laboratory of Gastric Tumor of Shanghai City, Shanghai 200025, China

This work was supported by the Chinese National High Tech Program (No. 2012AA02A504 and 2012AA02A203), National Natural Science Foundation of China (No. 81172329 and 81372644), Innovation Foundation of Translational Medicine of Shanghai Jiao Tong University School of Medicine (No. 15ZH1002 and 15ZH4001), and Project of Biobank of Gastrointestinal Carcinoma of Shanghai Jiao Tong University School of Medicine

Abstract Objective: To explore the quality of inventory samples of a biobank stored in a deep freezer from 0 to over 10 years in Shanghai Ruijin Hospital. Methods: We extracted 24 pairs of stocked gastric cancer samples between 2003 and 2014. We used 1% agarose 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 Coomassie 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 lowmolecular-weight (average 36.5 KD) proteins; 6-8 years, medium-molecular-weight (average 65.63KD) proteins; 3-5 years, high-molecular-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; likewise, degradation of proteins with higher molecular weight should occur.

Keywords:biosample, gastric cancer, cryo preservation, nucleic acid, protein, quality control

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