[1]罗 凯,黎谢梦丹,王 倩,等.Hsa-miR-17基因启动子区PCR扩增及鉴定体系的构建[J].现代检验医学杂志,2017,32(05):4-7,12.[doi:10.3969/j.issn.1671-7414.2017.05.002]
　LUO Kai,LIXIE Meng-dan,WANG Qian,et al.Establishment of the PCR Amplification and Sequencing System of Hsa-miR-17 Gene Promoter Region[J].Journal of Modern Laboratory Medicine,2017,32(05):4-7,12.[doi:10.3969/j.issn.1671-7414.2017.05.002]
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Hsa-miR-17基因启动子区PCR扩增及鉴定体系的构建()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第32卷
期数:
2017年05期
页码:
4-7,12
栏目:
论著
出版日期:
2017-11-02

文章信息/Info

Title:
Establishment of the PCR Amplification and Sequencing System of Hsa-miR-17 Gene Promoter Region
文章编号:
1671-7414(2017)05-004-05
作者:
罗 凯; 黎谢梦丹; 王 倩; 吴顺芳; 贾小婷; 贺智敏
广州医科大学附属肿瘤医院/广州医科大学肿瘤研究所,广州 510095
Author(s):
LUO Kai; LIXIE Meng-dan; WANG Qian; WU Shun-fang; JIA Xiao-ting; HE Zhi-min
Affiliated Cancer Hospital of Guangzhou Medical University,CancerResearch Institute of Guangzhou Medical University,Guangzhou 510095,China
关键词:
Has-miR-17;  基因;  PCR;  启动子
分类号:
R531.3; R381
DOI:
10.3969/j.issn.1671-7414.2017.05.002
文献标志码:
A
摘要:
目的 建立Has-miR-17编码基因启动子区的PCR扩增及鉴定体系。方法 提取人源细胞系基因组DNA为实验样本,以Has-miR-17编码基因上游启动子区序列为目标序列,行序列特征分析并设计特异PCR扩增及测序引物,利用添加DMSO和使用5'加尾引物的体系优化策略建立Has-miR-17基因启动子区PCR扩增及鉴定体系并行性能评估。同时另以10例人源细胞系为样本初步验证该PCR及鉴定体系的应用效果。结果 经电泳鉴定与测序分析确认在联合使用5'加尾PCR引物和5%DMSO的条件下Has-miR-17基因启动子区PCR扩增及鉴定体系已成功建立,其检测下限达10 ng/μl且特异度好、重复性佳。利用该体系成功实现对10例人源细胞株Has-miR-17基因启动子区的PCR扩增及鉴定。结论 已成功建立Has-miR-17编码基因启动子区的PCR扩增及鉴定体系。
Abstract:
Abstract:Objective To establish the PCR amplification and sequencing system of Hsa-miR-17 gene promoter region.Methods To establish the PCR amplification and sequencing system of Hsa-miR-17 gene promoter region and evaluate its performance by analyzing the sequence characteristics,designing PCR and sequencing primers,extracting sample genome DNA from different human cell lines and using optimization strategy of adding DMSO to the final concentration of 5% and making use of 5' tailed PCR primers; Then the established system was further verified in 10 other cell line samples.Results The PCR amplification and sequencing system of hsa-miR-17 gene promoter region,which had good repeatability,good specificity and its detection limit was 10 ng/μl,was established successfully under the condition of using 5' tailed PCR primers and 5% DMSO and that was confirmed by electrophoresis analysis and Sanger sequencing.And fragment of Has-miR-17 gene promoter region in 10 human cell lines were successfully achieved and verified bythis system.Conclusion The PCR system of Hsa-miR-17 gene promoter region was established successfully.

参考文献/References:

[1] 许文剑.miRNA与肿瘤关系的研究进展[J].医学综述,2013,19(17):3120-3123. Xu WJ.Research progress of correlation between mi-RNA and cancer[J].Medical Recapitulate,2013,19(17):3120-3123.
[2] 周明玉,岳双磊,何文杰,等.microRNA-214在恶性肿瘤中的表达及相关性的研究进展[J].现代生物医学进展,2017,17(4):787-790. Zhou MY,Yue SL,He WJ,et al.The research progress of miRNA-214 expression in malignant tumor and relationship between miRNA-214 and malignant tumor[J].Progress in Modern Biomedicine,2017,17(4):787-790.
[3] 邱 进,刘 辉,热孜宛古丽·伊敏,等.MEF2C基因启动子区碱基突变对转录活性的影响[J].新疆医科大学学报,2017,40(5):606-611,616. Qiu J,Liu H,Reziwanguli YM,et al.Effect of base mutation in MEF2C promoter region on transcription activity[J].Journal of Xinjiang Medical University,2017,40(5):606-611,616.
[4] 李 璐,梁 辉,陈群蓉.鼻咽癌组织中SYK基因启动子区的甲基化分析[J].现代检验医学杂志,2015,30(2):39-41,45. Li L,Liang H,Chen QR.Methylation of SYK gene promoter region in nasopharyngeal carcinoma[J].Journal of Modern Laboratory Medicine,2015,30(2):39-41,45.
[5] 裴静娴,王月刚,张艺军,等.p53RFP基因启动子荧光素酶报告基因载体的构建及鉴定[J].广东医学,2017,38(2):170-172. Pei JX,Wang YG,Zhang YJ,et al.Construction and identification ofluciferase reporter plasmid containing p53RFP gene promoter[J].Guangdong Medical Journal,2017,38(2):170-172.
[6] 李星光,卢圣奎,王 帅.microRNA-17过表达对胶质瘤患者预后的影响[J].脑与神经疾病杂志,2016,24(3):168-171. Li XG,Lu SK,Wang S.Influence of expression level of miR-17 on prognosis of glioma patients[J].Journal of Brain and Nervous Diseases,2016,24(3):168-171.
[7] 周君纯,郑 丽,刘家华,等.以microRNA-17为靶点的反义核酸对白血病细胞K562的作用[J].白血病·淋巴瘤,2013,22(7):428-430,435. Zhou JC,Zheng L,Liu JH,et al.Effects of antisense oligonucleotides targeting microRNA-17 on human leukemic K562 cells[J].Journal of Leukemia& Lymphoma,2013,22(7):428-430,435.
[8] Dellago H,Bobbili MR,Grillari J.MicroRNA-17-5p:At the crossroads ofcancer and aging-a mini-review[J].Gerontology,2017,63(1):20-28.
[9] Liao XH,Xiang Y,Yu CX,et al.STAT3 is required for MiR-17-5p-mediated sensitization to chemotherapy-induced apoptosis in breast cancer cells[J].Oncotarget,2017,8(9):15763-15774.
[10] Gong J,He L,Ma J,et al.The relationship between miR-17-5p,miR-92a,and let-7b expression with non-small cell lung cancer targeted drug resistance[J].Journal of the Balkan Union of Oncology,2017,22(2):454-461.
[11] 王 倩,罗 凯.一种EGFR基因突变检测方法的建立及初步应用[J].重庆医学,2014,43(11):1351-1353,1356. Wang Q,Luo K.Establishment of a method for detecting EGFR gene mutations and its preliminary application[J].Chongqing Medicine,2014,43(11):1351-1353,1356.
[12] Pratyush DD,Tiwari S,Kumar A,et al.A new approach to touch down method using betaine as cosolvent for increased specificity and intensity of GC rich gene amplification[J].Gene,2012,497(2):269-272.
[13] Obradovic J,Jurisic V,Tosic M,et al.Optimizationof PCR conditions for amplification of GC-rich EGFR promoter sequence[J].Journal of Clinical Laboratory Analysis,2013,27(6):487-493.
[14] Strien J,Sanft J,Mall G.Enhancement of PCR amplification of moderate GC-containing and highly GC-rich DNA sequences[J].Mol Biotechnol,2013,54(3):1048-1054.
[15] Vandenbussche F,Lefebvre DJ,De Leeuw I,et al.Laboratory validation of two real-time RT-PCR methods with 5'-tailed primers for an enhanced detection of foot-and-mouth disease virus[J].Journal of Virological Methods,2017,246:90-94.
[16] 罗 凯,黎谢梦丹,石兴源,等.人CDK14基因表达荧光定量PCR检测方法的建立及初步应用[J].现代检验医学杂志,2017,32(2):26-29. Luo K,Lixie MD,Shi XY,et al.Establishment and preliminary application of the method for detecting expression of human CDK14 with real-time quantitative PCR[J].Journal of Modern Laboratory Medicine,2017,32(2):26-29.

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备注/Memo

备注/Memo:
基金项目:广州市卫计委医药卫生科技项目(No.20161A011086)。 作者简介:罗 凯(1977-),男,硕士,副主任技师,肺癌的靶向治疗耐药机制与分子靶向检测,E-mail:luokainan@126.com。 通讯作者:贺智敏(1956-),男,博士,研究员,肿瘤的发生及耐药机制,E-mail:hezhimin2005@yahoo.com。
更新日期/Last Update: 1900-01-01