摘要
目的:探讨低温冻存时间对生物大分子DNA、RNA及蛋白质的影响,观察经长期低温冻存的肿瘤样本是否仍可满足科研的需求。方法:选择上海交通大学医学院附属瑞金医院上海消化外科研究所肿瘤样本库内2002至2006年间收集的手术切除胃癌标本20对(包括肿瘤组织及配对正常组织),用于提取组织总蛋白质和核酸,二喹啉甲酸(BCA)法测定蛋白浓度,采用凝胶电泳鉴定RNA、DNA完整性。采用逆转录-聚合酶链反应(RT-PCR)法检测核转录因子-κB(NF-κB)亚单位P65的mRNA表达;蛋白印迹(Western blot)法检测NF-κB蛋白表达。另随机抽取46例胃癌标本,检测其NF-κBP65的单核苷酸多态性(SNP)位点变异。结果:所有抽提DNA的吸光度比值(A260/A280)均在1.7～2.1间,电泳条带清晰,可检测出SNP位点变异。与保存1～2年者相比,储存3年或以上的标本NF-κBP65 mRNA与蛋白表达均有下调(P=0.03,P<0.01)。结论:组织样本低温保存在5年范围内,其DNA分子的完整性与质量均未受影响;RNA与蛋白质分子在低温保存1～2年内尚不影响癌基因表达的分析结果,但低温保存3年或以上的标本中RNA及蛋白质均有不同程度降解。
Objective To explore whether the long-term frozen-preserved bio-specimen can meet the needs of research study and whether affect the activity of biological macromolecules. Methods Twenty pairs of gastric cancer specimens collected from 2002 to 2006 and preserved in tissue bank of Shanghai Ruijin Hospital were used for quality verification. Total protein and nucleic acids were extracted. BCA method was used for determination of concentration of protein. The integrity of RNA and DNA was examined by gel electrophoresis. RT-PCR and Western blot were used for the assessment of expression of NF-KB mRNA and protein, respectively. Forty-six specimens were tested for the single nucleotide polymorphisms （SNP）of NF-KB. Results The A 260/A280 ratios of DNA extracted from all specimens were 1.7-2.1. The eleetrophoresis demonstrated the integrity of DNA and SNP of NF-KB could be detected. The expressions of NF-kB mRNA and protein were higher in specimens stored for less than 2 years than that stored for no less than 3 years （P=0.03 and P〈0.01, respectively）. Conclusions The integrity of DNA molecules is not affected by long-term （5 years） frozen- preservation. Short-term （1-2 years） frozen- preservation does not affect the RNA and protein expressions in cancer gene study, but long-term preservation （3 years or more）could degrade the RNA and protein molecules in certain degree.
出处
《诊断学理论与实践》
2009年第1期38-42,共5页
Journal of Diagnostics Concepts & Practice
基金
国家科技部"863"重大专项资助(2006AA02A402
2006AA02A301)
国家自然科学基金项目(30572127
30770961
306709391)
上海市浦江人才计划项目(PJ200700367)
上海市科委重点基础研究项目(05JC14013)
关键词
肿瘤
标本
技术方法
癌基因
生物大分子
低温冻存
Neoplasms
Specimen
Technology
Oncogene
Biomacromolecule
Frozen preservation
