Moll Pamela R, Duschl Jutta, Richter Klaus
Fachbereich Zellbiologie, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
Anal Biochem. 2004 Nov 1;334(1):164-74. doi: 10.1016/j.ab.2004.07.013.
The use of expression profiling to explore a cell's transcriptional landscape has exploded in recent years. In many cases, however, the very limited amount of starting material poses a major problem, making the amplification of the isolated RNA obligatory. The most prominent amplification method used was developed by the Eberwine lab in 1990: cDNA synthesis is started with an oligo(dT) primer containing a T7 RNA polymerase promoter. After second-strand synthesis RNA is transcribed in vitro using T7 RNA polymerase. It has been demonstrated that antisense RNA amplification not only preserves the fidelity of RNA-based microarray analysis but even improves the sensitivity. In our aim to improve the yield of in vitro transcription reactions and to facilitate the use of amplified RNA for the construction of cDNA libraries we tested a series of T7 primers with different 3' flanking sequences containing restriction sites. In addition we tested the impact of different DNA polymerases used for synthesizing the templates on the efficiency of the in vitro transcription reaction. A total of 28 different oligo(dT)-T7 promoter primers were tested. Two of them showed a dramatically increased yield of RNA from the in vitro transcription reaction. The combination of the improved second-strand synthesis with the new T7 primer increased the RNA yield 60-fold compared to the yield of standard procedures.
近年来，利用表达谱分析来探索细胞转录图谱的研究呈爆发式增长。然而，在许多情况下，起始材料的量非常有限，这成为一个主要问题，使得对分离出的RNA进行扩增成为必需。最著名的扩增方法是由埃伯温实验室于1990年开发的：cDNA合成从含有T7 RNA聚合酶启动子的寡聚（dT）引物开始。在第二链合成后，使用T7 RNA聚合酶在体外转录RNA。已经证明，反义RNA扩增不仅能保持基于RNA的微阵列分析的保真度，甚至还能提高灵敏度。为了提高体外转录反应的产量，并便于将扩增的RNA用于构建cDNA文库，我们测试了一系列具有不同3'侧翼序列且含有限制性酶切位点的T7引物。此外，我们还测试了用于合成模板的不同DNA聚合酶对体外转录反应效率的影响。总共测试了28种不同的寡聚（dT）-T7启动子引物。其中两种引物在体外转录反应中显示出RNA产量显著提高。与标准程序相比，改进的第二链合成与新型T7引物的组合使RNA产量提高了60倍。
