中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3697-3700.doi: 10.3969/j.issn.1673-8225.2011.20.021

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

高效快速提取股骨头中总RNA的方法

柳  铭1，李章华1，陈  颖2，王  芳2，夏  伟2，陈友浩1，潘  峰1   

  1. 1 武汉大学人民医院骨一科，湖北省武汉市  430060
    2 解放军军事医学科学院基础医学研究所生物化学与分子生物学研究室，北京市  100850
  • 收稿日期:2011-02-28 修回日期:2011-03-26 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 李章华，博士，副主任医师，硕士生导师，武汉大学人民医院骨一科，湖北省武汉市 430060 lzh999999@ yahoo.com.cn
  • 作者简介:柳铭★，男，1986年生，湖北省潜江市人，汉族，武汉大学在读硕士，主要从事股骨头坏死过程中相关基因表达变化的研究。 wang52736lm@163.com
  • 基金资助:

    国家自然科学基金(30700854，81071463)，课题名称：cbfa1基因促进低氧状态下间充质干细胞生物学特性的实验研究。湖北省自然科学基金(2009CDB414)。

Efficient and quick method of extracting total RNA from the femoral head

Liu Ming1, Li Zhang-hua1, Chen Ying2, Wang Fang2, Xia Wei2, Chen You-hao1, Pan Feng1   

  1. 1Department of Orthopaedics, Renmin Hospital of Wuhan University, Wuhan  430060, Hubei Province, China
    2Department of Biochemistry and Molecular Biology, Insititute of Basic Medical Science, Academy of Military Sciences, Beijing  100085, China 
  • Received:2011-02-28 Revised:2011-03-26 Online:2011-05-14 Published:2011-05-14
  • Contact: Li Zhang-hua, Doctor, Associate chief physician, Master’s supervisor, Department of Orthopaedics, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China lzh999999@yahoo. com.cn
  • About author:Liu Ming★, Studying for master’s degree, Department of Orthopaedics, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China wang52736lm@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30700854*, 81071463*; the Natural Science Foundation of Hubei Province, No. 2009CDB414*

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683

被引次数

3

摘要：

背景：从骨组织中有效提取总RNA是进行骨科相关实验的基础，目前所知方法的提取效果不理想。
目的：探寻一种高效、快速的骨组织总RNA提取方法。
方法：取健康大耳白兔股骨头迅速置于用液氮预冷过的研钵中，液氮中反复研磨至粉末状，再将其移至预冷的匀浆器内，加入Trizol，充分匀浆后4 ℃离心，取上清，加入氯仿离心，使RNA与细胞DNA、蛋白质及其他成分分离从而得到总RNA。另取兔股骨头，按传统方法取材后保存，无匀浆过程，传统Trizol法提取总RNA作为对照。紫外分光光度计测定RNA样品浓度、纯度及产率。甲醛变性胶电泳观察RNA的28 S，18 S条带是否清晰，有无降解和DNA污染。
结果与结论：实验方法提取的总RNA浓度在0.80~0.90 g/L，A260/A280在1.90~2.00之间，A260/A230在1.4~1.6之间，明显高于传统方法提取的RNA各指标，说明实验方法提取的总RNA纯度高，无DNA、蛋白质污染。甲醛变性胶电泳可清晰显示28 S，18 S两条带，大体观察其比例大约为1∶1，证实RNA提取完整，没有降解。由此可见，实验方法提取的骨组织总RNA质量高，提取方法方便、快捷，可用于骨组织分子生物学研究。

关键词: 股骨头, 抽提, 总RNA, 方法, 组织工程

Abstract:

BACKGROUND: There are no ideal methods to extract total RNA from bone tissues.
OBJECTIVE: To explore an efficient and quick method of acquiring total RNA from bone tissue.
METHODS: Ten healthy rabbits were divided into experimental group and control group on average. The femoral heads of experimental group were removed by rongeur which has been disinfected, then stored in liquid nitrogen after quick-freeze. Normal way was used to get control group’s femoral heads. Experimental group’s femoral head was immediately placed in mortar that has been precooled by liquid nitrogen. The bone was grinded iteratively in mortar until it became bone powder, the powder was transferred into a homogenizer which has been precooled, added Trizol, centrifuged at 4 ℃ after fully homogenized to get supernatant. Then chloroform and other organic solvents were added, centrifuged, and then the total RNA was separated from DNA, protein and other tissues. Control group’s RNA was extracted by traditional Trizols method. The concentration, purity and productive rate were measured by ultraviolet spectrophotometer. Finally, denaturing agarose-formalhyde gel electrophoresis was used to observe if the two bands (28 S,18 S) of RNA were clear, RNA was degradated and with/without DNA contamination.
RESULTS AND CONCLUSION: The extracted RNA were in high purity without DNA and protein contamination; The result of degenerated formaldehyde electrophoresis shows that the 28 S and 18 S bands were clear and the ratio of them was about 1:1, which confirmed that RNA was complete with no degradation. The findings from the present study show that this method is a rapid and efficient purification method for gaining total RNA form bone tissue, and it can be used for analyzing molecular biology of bone tissue.

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