双荧光标记法定量检测Igf-2r在克隆牛组织中的表达

Quantitative detection of Igf-2r in cloned cattle with dipl-fluorescent labeling real-time PCR

目的建立克隆牛Igf-2rmRNA实时定量PCR的检测方法,并探讨Igf-2rmRNA的表达在克隆牛发育中的作用。方法用Trizol抽提正常及克隆牛的脑、肺、心、肝组织RNA,运用本研究建立的实时荧光定量PCR方法检测各组织中Igf-2rmRNA的表达情况。结果正常牛各组织中每μg总RNA中含Igf-2rmRNA拷贝数分别为107.39(脑)、108.04(肺)、107.25(心)、107.99(肝)。克隆牛中,每μg总RNA中含Igf-2rmRNA表达水平变化较大。与正常牛相比,克隆牛不同组织中Igf-2rmRNA的表达水平变化较大,在同一克隆牛的不同组织以及不同克隆牛的同一组织中Igf-2rmRNA的表达水平相差较大,提示在克隆牛不同组织中Igf-2rmRNA的表达水平不一。结论Igf-2rmRNA的表达水平与动物的生长发育有一定的关系,Igf-2r基因的表达异常可能与胎牛过度肥大和围产期死亡等现象有一定的关系。对克隆牛各组织中Igf-2rmRNA的表达的精确定量有助于进一步研究Igf-2r基因在动物生长发育中所起的作用。

Objective To established a method of real-time PCR to quantized Igf-2r mRNA in cloned cattle and to discuss the mechanism of Igf-2r mRNA express in cloned cattle. Methods We extracted tissues（brain, lung, heart, liver） mRNA of normal and cloned cattle with Trizol by using the method of real-time fluorescent quantitative PCR（FQ-PCR）, and quantized Igf-2r mRNA of tissues in cloned cattle. Results In normal cattle, the Igf-2r mRNA in brain, lung, heart, liver were 10^7.39 , 10^8.04,10^7.25, 10^7.99 copies per μg, respectively. In cloned cattle the copies of Igf-2r mRNA were variable. Compared with the normal cattle, the express levels of Igf-2r mRNA of different tissues in cloned cattle were various. Conclusions The express level of Igf-2r mRNA may relative with the growth and development of some animals, the abnormality express level of Igf-2r may result in disproportional overgrowth and perinatal death of animals. Precise quantitation of the Igf-2r mRNA in cloned cattle may conduce to study the effect of Igf-2r in the growth and development of animals.