荧光定量聚合酶链反应法比较标准管家基因3-磷酸甘油醛脱氢酶、β-肌动蛋白、酸性核糖体磷蛋白P0及18S核糖体RNA在老年大鼠不同组织中的表达

Comparison of standard housekeeping genes G3pd, ACTB, Arbp and 18S rRNA expression levels in aging rat tissues by real-time fluorescent polymerase chain reaction

背景:反转录聚合酶链反应用于基因表达分析越来越显示出其重要性,其中合适的内参基因选择很重要,目前并没有适合于所有体系可作为内参的管家基因。大鼠是常用动物模型,选择合适的大鼠管家基因作为内参尤其必要。目的:比较分析使用最广泛的4个标准管家基因3-磷酸甘油醛脱氢酶、β-肌动蛋白、酸性核糖体磷蛋白P0及18S核糖体RNA在老年大鼠肝、肾、心、肺组织和大脑皮质中的表达情况。设计、时间及地点:观察对比实验,于2007-07/08在泰山医学院生命科学研究所分子生物学实验室及泰安市疾控中心病毒检测中心实验室完成。材料:选用老年SD大鼠3只,用于检测管家基因表达情况。方法:采用反转录实时荧光聚合酶链反应技术检测老年大鼠肝、肾、心、肺组织和大脑皮质中有着不同表达丰度和功能的4个管家基因3-磷酸甘油醛脱氢酶、β-肌动蛋白、酸性核糖体磷蛋白P0及18S核糖体RNA的表达情况。主要观察指标:老年大鼠不同组织内4个管家基因的表达水平及不同组织间的表达差异。结果:①酸性核糖体磷蛋白P0在老年大鼠不同组织间表达差异最小。②老年大鼠肾组织中表达最稳定的管家基因是β-肌动蛋白;心脏组织和肺组织中表达最稳定的管家基因是3-磷酸甘油醛脱氢酶。结论:老年大鼠组织间信使RNA分析仅用1个内参基因是不合适的,至少应该使用两个参照基因,1个选用18S核糖体RNA;另一个基因的选择标准如下:适用于分析不同组织间基因表达情况的内参基因是酸性核糖体磷蛋白P0,而心脏和肺组织研究适用3-磷酸甘油醛脱氢酶,肾组织研究适用β-肌动蛋白。

BACKGROUND： Reverse transcription-polymerase chain reaction has become more and more important in gene expression analysis. Selection of proper reference gene is very important. At present, there is no one absolute housekeeping gene appropriate to all experiments as inner control. Selection of proper rat housekeeping genes as inner control is particularly necessary as a rat model is frequently used. OBJECTIVE： To compare the expression levels of the 4 most widely used standard housekeeping genes glyceraldehyde-3-phosphate dehydrogenase（G3pd）, β-actin（ACTB）, acidic ribosomal phosphoprotein P0 （Arbp） and 18S ribosomal RNA （18S rRNA ） in liver, kidney, heart, lung and cortex of aging rats. DESIGN, TIME AND SETTING： Observative and comparison experiment, the experiment was performed at Molecular Biology Laboratory of Institute of Life Science, Taishan Medical University and Virus Detection Center Laboratory, Taian Municipal Center for Disease Control ＆ Prevention between July 2007 and August 2007. MATERIALS： Three aging SD rats were selected to detect the expression levels of housekeeping genes. METHODS： Real-time reverse transcription-polymerase chain reaction was used to examine the expression levels of the four housekeeping genes with different abundances and functions： G3pd, ACTB, Arbp and 18S rRNA in aging rat liver, kidney, heart, lung and cortex. MAIN OUTCOME MEASURES： The expression levels of 4 housekeeping genes in aging rat different tissues and differences in the expression levels among different tissues. RESULTS： The Arbp gene expression level varied minimally among different aging rat tissues. The housekeeping gene expressed most stably was ACTB in aging rat kidney and was G3pd in heart and lung. CONCLUSION： It is improper to use only one reference gene for mRNA analysis, and at least two housekeeping genes should be used. One is the 18S rRNA and the other proper inner control gene is Arbp for aging rat inter-tissue comparison. As for intra-tissue comparison, G3pd should be considered for heart or lung and ACTB for kidney.