摘要
背景:研究表明叉头转录因子1是一种新的2型糖尿病易感基因,但2型糖尿病患者与健康人的叉头转录因子1基因在转录水平上差异性的研究不多。由于还没有商品化叉头转录因子1的质粒标准品,因此有必要构建其标准品。目的:构建应用实时荧光定量-聚合酶链反应检测叉头转录因子1mRNA的质粒标准品。设计、时间及地点:单一样本重复实验,于2007-11/2008-03在苏州大学附属第一医院检验科实验室完成。材料:SYBRExScriptTM反转录-聚合酶链反应Kit试剂盒,SYBRPremixExTaqTM试剂盒,凝胶回收纯化DNA试剂盒,质粒DNA小量纯化试剂盒,PMD18-TVector试剂盒等。方法:从健康志愿者血标本中提取总RNA,反转录合成cDNA,以cDNA为模板应用聚合酶链反应扩增出目的片段,对其回收纯化连接到PMD18-T载体及转化JM109,阳性克隆经测序分析,确认重组质粒正确。抽提重组质粒,用紫外分光光度仪测定其浓度,再根据公式换算成拷贝浓度,最后稀释成梯度标准品。主要观察指标:重组质粒聚合酶链反应和基因测序结果,标准品标准曲线的相关系数及标准品的重复性。结果:成功构建了叉头转录因子1质粒标准品,通过聚合酶链反应及基因测序鉴定其目的片段已成功插入载体。各稀释度的标准品经荧光定量-聚合酶链反应扩增后,其循环阈值与起始模板量的对数值之间有着良好的线性关系,线性范围是2.39×(105～1012)copies/mL。各稀释度标准品的批内重复性好,其变异系数范围为0.29%～1.16%。结论:实验成功构建了具有较好灵敏度和重复性的叉头转录因子1质粒标准品。
BACKGROUND： Studies show that forkhead transcription factor 1 （Fox01） plays an important role in insulin resistance and the pathogenesis of type 2 diabetes. However, research regarding diversity of normal Fox01 and diabetics are few. Therefore, it is necessary to construct the plasmid standard. OBJECTIVE： To construct the plasmids standard of fox01 mRNA, in addition, to detect the standard by the method of RT-PCR. DESIGN, TIME AND SETTING： The experiment of repeating single sample was performed at the First Affiliated Hospital of Soochow University Laboratory from November 2007 to March 2008. MATERIALS： SYBR ExScriptTMRT-PCR Kit, SYBR Premix Ex TaqTM kits, TaKaRa agarose gel DNA purification kit, TaKaRa Minibest plasmid DNA purification kit, PMD18-T Vector Kit, and other reagents, primers and equipments. METHODS： The total RNA was extracted from the blood samples of healthy volunteers. Then cDNA was synthesized by reverse transcription. The target sequence was amplified from cDNA, and then the PCR product was gel extracted and purified. Products of purification were connected to PMD18-T vector and transformed into competent cells JM109. The method of sequencing was used to verify that the target fragments were transformed successfully. Plasmid DNA was extracted and was measured using UV spectrophotometer and converted to copies/mL according the equation. Finally it was diluted into a standard gradient. MAIN OUTCOME MEASURES： The results of recombinant plasmid PCR and gene sequencing, the correlation coefficient of the standard curve and the repeatability of the plasmid standard. RESULTS： A plasmid standard for Fox01 was successfully constructed. The methods of sequencing and PCR verified that the target fragments were transformed successfully. After amplified by FQ-PCR, the standards of the different concentration had good linear relationship between the cycle threshold and the numerical of the start template. The linear range was 2.39x （105-1012） copies/mE The standards of the different con
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第7期1263-1266,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江苏省一三五临床免疫学重点实验室高新技术平台研究课题~~
