中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (11): 1973-1976.doi: 10.3969/j.issn.1673-8225.2012.11.017

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

石蜡包埋组织提取DNA不同条件下的PCR扩增★

马丽丽,艾力江•吐尔逊,张  莉   

  1. 新疆医科大学第一附属医院肿瘤中心,新疆维吾尔自治区乌鲁木齐市  830011
  • 收稿日期:2012-01-05 修回日期:2012-02-02 出版日期:2012-03-11
  • 通讯作者: 张莉,博士生导师,教授,主任医师,新疆医科大学第一附属医院肿瘤中心,新疆维吾尔自治区乌鲁木齐市 830011 zhangli9514@126. com
  • 作者简介:马丽丽★,女,1984年生,新疆维吾尔自治区乌鲁木齐市人,回族,新疆医科大学在读硕士,主要从事食管癌的个体化治疗研究。1215147959@qq.com

PCR amplication of the DNA extracted from paraffin-embedded tissues in different conditions

Ma Li-li, Ailijiang•Tuerxun, Zhang Li   

  1. Cancer Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi    830011, Xinjiang Uygur Autonomous Region, China
  • Received:2012-01-05 Revised:2012-02-02 Online:2012-03-11
  • Contact: author: Zhang Li, Doctoral supervisor, Professor, Chief physician, Cancer Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China Zhangli9514@126.com
  • About author:Ma Li-li★, Studying for master’s degree, Cancer Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China 1215147959@qq.com

摘要:

背景:在甲醛固定石蜡包埋组织的制作及保存过程中会对DNA造成损害,提取的DNA在用于PCR扩增时部分结果并不理想。
目的:比较分析石蜡包埋组织中提取DNA应用于PCR反应的影响因素。
方法:从10.0 μm×2、10.0 μm×4和10.0 μm×5石蜡包埋食管癌组织切片中提取DNA,以0.05,0.1,0.2 μg模板DNA量扩增内参基因β-actin,PCR反应循环次数分别为35,40,45次,分析影响PCR反应的因素。
结果与结论:琼脂糖凝胶电泳结果显示以10.0 μm×2组织切片量,PCR反应循环次数40次,PCR反应模板DNA量      0.05 μg扩增取得的目的基因DNA的质量最高。说明减少石蜡包埋组织用量和减少模板DNA量有助于获得高质量的PCR反应条带,且PCR反应循环次数应大于40次,小于45次,再增加循环次数,则意义不大。
关键词:石蜡包埋组织;提取DNA;PCR反应;循环次数;模板DNA
doi:10.3969/j.issn.1673-8225.2012.11.017

关键词: 石蜡包埋组织, 提取DNA, PCR反应, 循环次数, 模板DNA

Abstract:

BACKGROUND: Because of the damage of formalin-fixed paraffin-embedded tissue to DNA in the process of its production and preservation, it is difficult to extract high quality and sufficient DNA for PCR.
OBJECTIVE: To analyze the influencing factors of the DNA extracted from paraffin-embedded tissue for PCR.
METHODS: Genomic DNA was extracted from paraffin-embedded esophageal carcinoma tissue sections of 10.0 μm×2,      10.0 μm×4 and 10.0 μm×5. Template DNA amount of 0.05, 0.1, 0.2 μg was used to amplify gene β-actin, respectively. PCR cycle times were set as 35, 40 and 45. The influencing factors of PCR were analyzed.
RESULTS AND CONCLUSION: The analysis of agarose gel electrophoresis showed that when paraffin-embedded esophageal carcinoma tissue section amount was 10.0 μm×2, the PCR cycle times were 40; when the template DNA amount was 0.05μg, the obtained target DNA was of the highest quality. It shows that reducing the amount of paraffin-embedded tissue and DNA template can be help to require high quality PCR strips; PCR cycle times should be more than 40 and less than 45, and if it increases beyond this range there is meaningless.

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