应用Super SMART cDNA合成技术扩增哮喘嗜酸性粒细胞总RNA

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (09): 1037-1039.

应用Super SMART cDNA合成技术扩增哮喘嗜酸性粒细胞总RNA

赵海金¹, 蔡绍曦¹, 邹飞², 佟万成¹, 万为人²

1. 第一军医大学, 南方医院呼吸科, 广东, 广州, 510515;
2. 第一军医大学, 热带军队卫生学系高温医学研究室, 广东, 广州, 510515

出版日期:2004-09-20 发布日期:2004-09-20
基金资助:
收稿日期:2004-3-16。
基金项目:国家自然科学基金(30270593)
作者简介:赵海金(1975-),男,第一军医大学在读硕士生,主治医生,电话:020-61641575
通讯作者:蔡绍曦,电话:020-61641571

Super SMART cDNA synthesis technology for amplifying small amount of total RNA of peripheral blood eosinophils from asthma patients

ZHAO Hai-jin¹, CAI Shao-xi¹, ZOU Fei², TONG Wan-cheng¹, WAN Wei-ren²

1. 第一军医大学, 南方医院呼吸科, 广东, 广州, 510515;
2. 第一军医大学, 热带军队卫生学系高温医学研究室, 广东, 广州, 510515

Online:2004-09-20 Published:2004-09-20

摘要/Abstract

摘要： 目的应用Super SMART cDNA 合成技术从嗜酸细胞微量总RNA扩增大量双链cDNA。方法利用Percoll密度梯度离心分离哮喘病人血嗜酸性粒细胞,用Trizol试剂盒提取细胞总RNA,利用Super SMART cDNA合成技术合成cDNA第1链及优化扩增第2链。通过梯度电泳鉴定cDNA产品质量,通过对获得的cDNA定量来评估扩增效能。结果从20 ng总RNA成功扩增出双链cDNA 7.155 μg,电泳结果显示cDNA质量及纯度较高。结论 Super Smart cDNA 合成技术可实现从微量总RNA高效扩增高质量的cDNA,为进一步从基因水平研究哮喘机制奠定了基础。

Abstract: Objective To amplify double-strand cDNA from small amount of total RNA of eosinophils from asthma patients by Super SMART cDNA synthesis technique. Methods The eosinophils were purified from the peripheral blood of asthma patients before and after treatment by Percoll gradient centrifugation, from which the total RNA was extracted using TRIzol kit. First-strand cDNA synthesis and double-strand cDNA amplification were performed using Super SMART cDNA synthesis technique. The quality of the obtained cDNA was evaluated by gradient cDNA electrophoresis, and the amplification efficiency determined by cDNA quantification. Result From 20 ng total RNA, 7.155 μg and 6.568 μg of the tester and driver double- strand cDNAs respectively were obtained successfully, and the result of electrophoresis indicated high quality and purity of the cDNA acquired. Conclusion Super SMART cDNA synthesis technique can effectively amplify high-quality double-strand DNA from a very small amount of total RNA, which may facilitate the exploration of the mechanism of asthma from the genetic level.

中图分类号:

Q786

赵海金¹, 蔡绍曦¹, 邹飞², 佟万成¹, 万为人². 应用Super SMART cDNA合成技术扩增哮喘嗜酸性粒细胞总RNA[J]. 南方医科大学学报, 2004, 24(09): 1037-1039.

ZHAO Hai-jin¹, CAI Shao-xi¹, ZOU Fei², TONG Wan-cheng¹, WAN Wei-ren². Super SMART cDNA synthesis technology for amplifying small amount of total RNA of peripheral blood eosinophils from asthma patients[J]. Journal of Southern Medical University, 2004, 24(09): 1037-1039.

参考文献

[1] 罗亮,岁雅玲,文岩.火把花根片对哮喘豚鼠肺泡灌洗液白细胞介素3受体mRNA的表达及嗜酸性粒细胞浸润的影响[J].第一军医大学学报,2003,23(10):1069-73.Luo L, Luo YL, Wen Y. Effects of Huobahuagen tablet on the expression of interleukin-3 receptor mRNA in asthmatic guinea pig bronchoalveolar lavage fluid and eosinophil infiltration[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(10): 1069-73.
[2] 徐劲松,蔡绍曦,邹飞,等.应用抑制消减杂交克隆支气管哮喘病人嗜酸细胞差异表达基因[J].第一军医大学学报,2004,24(5):509-12Xu JS, Cai SX, Zou F, et al. Cloning of differentially expressed genes of eosinophils from asthmatic patients by suppression subtractive hybridization[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(5):509-12.
[3] 中华医学会呼吸病学分会哮喘学组.支气管哮喘防治指南(支气管哮喘的定义、诊断、治疗及教育和管理方案)[J].中华结核和呼吸杂(Chin J Tuberc Respir Dis),2003,26(3):132-8.
[4] Zhu YY, Machleder EM, Chenchik A, et al. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction[J]. Biotechniques, 2001, 30(4): 892-7.
[5] Lukyanov K, Diatchenko L, Chenchik A, et al. Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect[J]. Biochem Biophys Res Commun, 1997, 230(2): 285-8.
[6] 杨昆,黄绍光.支气管哮喘流行变化趋势分析[J].中华结核和呼吸杂志(Chin JTuberc RespirDis),2001,24(3):181-3.
[7] Oh JH, Kim YS, Kim NS. An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material[J]. Exp Mol Med, 2003, 35(6): 586-90.
[8] Reddy MK, Nair S, Sopory SK. Global amplification ofcDNA from limiting amounts of tissue, an improved method for gene cloning and analysis[J]. Mol Biotechnol, 2002, 22(3): 223-34.

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