非同位素mRNA差异显示方法的建立及应用

Differential Display of mRNAs by a PCR-based Method Without the Use of Radioisotopes

目的建立非同位素mRNA差异显示技术 ,并用以分析鼠脑甲状腺激素的反应基因。 方法用 9-1 0bp的随机但序列确定的引物扩增甲减和正常大鼠脑cDNA ,用PAGE分析PCR产物 ,EB染色 ,切取差异条带 ,再扩增、测序并作同源性分析。 结果得到两个 (2 2 6、2 79bp)受甲状腺激素上调、一个 (2 78bp)受甲状腺激素下调的片段 ,前二者与任何已知基因的同源性都低于 80 % ,提示为两个尚未报道的新基因 ,后者即大鼠G蛋白Go的α亚单位基因。 结论非同位素mRNA差异显示技术是一种快速、简便。

Objective To develop a PCR-based method of differential display of mRNAs without the use of radioisotopes. Methods The cDNAs from normal and hypothyroid rats were amplified by using two 9-10bp primers arbitrarily with but defined sequence. The PCR products were analyzed by 8% PAGE and stained with EB. The bands showed significant difference between the normal and hypothyroid were cut out for reamplification. The reamplification products were subcloned into pGEM-T easy vector and sequenced. Results Three fragments were obtained, two down-regulated in hypothyroidism and one up-regulated in hypothyroidism. DNA sequencing analysis revealed that the two clones appearing lowly expressed in hypothyroidism showed no homology to known sequences in the GenBank, while the another clone appearing to highly expressed in hypothyroidism was identical to the α- subunit of rat G protein Go. Conclusion The present protocol of differential display of mRNAs without the use of radioisotopes is a rapid, simple and sensitive method to identify differentially expressed genes.