[1]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(02):26-29.[doi:10.3969/j.issn.1671-7414.2017.02.007]
　LUO Kai,LI Xie-meng-dan,SHI Xing-yuan,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(02):26-29.[doi:10.3969/j.issn.1671-7414.2017.02.007]
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人CDK14基因表达荧光定量PCR检测方法的建立及初步应用()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第32卷
期数:
2017年02期
页码:
26-29
栏目:
论著
出版日期:
2017-03-25

文章信息/Info

Title:
Establishment and Preliminary Application of the Method for Detecting Expression of Human CDK14 with Real-Time Quantitative PCR
文章编号:
1671-7414(2017)02-026-04
作者:
罗 凯; 黎谢梦丹; 石兴源; 贾晓婷; 王 倩; 邓 敏; 仇秦威; 张志杰; 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; LI Xie-meng-dan; SHI Xing-yuan; JIA Xiao-ting; WANG Qian; DENG Min;  ZHANG Zhi-jie; HE Zhi-min
Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Research Institute of Guangzhou Medical University,Guangzhou 510095,China
关键词:
细胞周期蛋白依赖性激酶14;  基因;  实时荧光定量PCR
分类号:
Q503
DOI:
10.3969/j.issn.1671-7414.2017.02.007
文献标志码:
A
摘要:
目的 建立一种人细胞周期蛋白依赖性激酶14(CDK14)基因表达荧光定量PCR检测方法。方法 利用Trizol法提取不同人肺癌细胞株中总RNA,以β-Actin和CDK14分别为内参和目的基因,分别设计特异性扩增引物,建立人CDK14基因表达荧光定量PCR检测系统并评估其检测性能。同时将其应用于不同细胞系siRNA干扰前后的CDK14基因相对表达水平检测。结果 经电泳分析、熔解曲线、产物测序确认人CDK14基因表达荧光定量PCR检测系统已建立,其特异性良好,重复性佳(CV=7.13%),线性范围较宽(CDK14与β-Actin分别在Ct值范围22.47～32.96与15.14～27.55间具有良好的相关性,r2=0.9844。)。利用该体系检测发现:在HCC827 D5和H1650细胞中CDK14基因高表达; 在1～3号干扰片段中,1号片段为有效片段。结论 人CDK14基因表达荧光定量PCR检测方法已成功建立。
Abstract:
Objective The method for detecting expression of human CDK14 gene with Real-time quantitative PCR was developed.Methods To establish a method for detecting expression of human CDK14gene with Real-time quantitative PCR by designing and synthesis of the primersof CDK14 target gene and β-Actin reference gene and extracting total RNA fromdifferent lung cancer cell lines.Then the specificity,detection range and repeatability of this method were evaluated.At last,the expression level of CDK14gene in different cell lines,which were with or without siRNA interference,were carried out by using this method.Results The method for detecting expression of human CDK14 gene with Real-time quantitative PCR,which had good specificity,good repeatability(CV=7.3%)and wide detection range(Ct value range of CDK14 and β-Actin amplification curve were 22.47～32.96 and 15.14～27.55 respectively,r2=0.9844),was developed and it was verified by electrophoresis analysis,melting curve,PCR product sequencing.And CDK14 gene expression level,which was detected by this method,increased in HCC827 D5,H1650 and number 1 siRNA segment was effective interference segment.Conclusion The method for detecting expression of human CDK14 gene with Real-time quantitative PCR was established successfully.

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备注/Memo

备注/Memo:
基金项目:广东省中医药局科研项目(No.20161179),广州市卫计委医药卫生科技项目(No.20161A011086)。
作者简介:罗 凯(1970-),男,博士研究生,副主任技师,研究方向:肺癌的靶向治疗耐药机制与分子靶向检测,E-mail:luolainan@126.com。
通讯作者:张志杰(1978-),男,博士研究生,助理研究员,研究方向:肿瘤的发生机制,E-mail:303589586@qq.com。
更新日期/Last Update: 2017-04-10