两种微量RNA扩增方法的比较研究  

Comparison of Two Different Amplification Methods for Small Amounts of Total RNA

作　　者：陈林华[1] 赵月娥[1] 陈晓燕[1] 高英[1] 

机构地区：[1]温州医学院附属眼视光医院,温州325027

出　　处：《中国细胞生物学学报》2011年第4期391-396,共6页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(No.30970924);浙江省自然科学基金(No.Y2100855);温州医学院附属眼视光医院科研启动基金(No.KYQD090701)资助项目~~

摘　　要：本研究通过比较体外转录和单引物扩增这两种扩增微量RNA的不同方法,以寻找一种高效的扩增方法。我们用两种不同方法分别扩增小鼠大脑全皮层及第五皮层细胞的RNA,扩增的RNA合成cDNA后进行荧光定量PCR实验,根据PCR结果比较两种不同扩增方法的效率。WT-ovation扩增RNA的效率约为IVT效率的2.8倍;IVT方法扩增后,基因D-C值与引物距离mRNA 3'端的长度及mRNA的长度均存在正线性相关(P<0.05),即引物距离mRNA的3'端越近、mRNA越短,基因D-Ct值越低。而WT-ovation方法扩增后,基因D-Ct值与引物距离mRNA 3'端的长度及mRNA的长度均不存在统计学相关性。与IVT方法相比,WT-ovation方法效率更高,扩增时受影响因素较少、更稳定。Two RNA amplification methods of in vitro transcription （IVT） and single primer amplification （WT-ovation） were compared to find a more efficient method for amplification of small amounts of total RNA. RNA was extracted from mouse cerebral cortex tissue or the layer V of cortical cortex, and then was amplified separately with the two different methods. After cDNA was reverse transcripted from the amplified RNA, real-time quantitative PCR was performed. The efficiency of RNA amplification was determined according to the results of real-time PCR. Our results indicated that WT-ovation amplification was about 2.8 times more efficient than IVT amplification. The value of D-Ct was positively correlated with both the primer length from the 3＇ terminus of mRNA（P〈0.05） and mRNA length（P〈0.05） after IVT amplification. The nearer distance between primer and the 3＇ terminus of mRNA and the shorter mRNA could result in lower value of D-Ct. But there was no significant correlation between the value of D-Ct and the primer position or mRNA length when the amplification method of WT- ovation was performed. Compared with IVT, WT-ovation was a more stable method which was more efficient and less affected by amplification factors.

关 键 词：RNA扩增 IVT扩增 WT-ovation扩增 定量PCR 

分 类 号：Q503[生物学—生物化学]