Bak Mads, Conley Lene, Hedegaard Jakob, Larsen Lars Allan, Sørensen Peter, Bendixen Christian, Tommerup Niels
Wilhelm Johannsen Center for Functional Genome Research, Institute of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, DK-2200N Copenhagen, Denmark.
Anal Biochem. 2006 Nov 1;358(1):111-9. doi: 10.1016/j.ab.2006.08.023. Epub 2006 Sep 7.
Several methods have been developed for amplification of RNA, making it possible to use cDNA microarrays for analysis of samples limited in amount of total RNA. The most widely used amplification protocol, the Eberwine method, amplifies RNA in a linear manner through in vitro transcription (IVT). However, when starting material is limited to nanogram amounts of total RNA, several rounds of amplification are necessary, making this method both expensive and labor-intensive. Amplification by PCR is robust and is able to amplify extremely limiting material. However, it is possible that the nonlinear nature of PCR could result in reduced reproducibility of the amplification compared with IVT. We have evaluated two methods that use a combination of PCR and IVT for amplification of nanogram amounts of total RNA. We have compared microarray results obtained by these methods with results obtained by two established methods: indirect labeling of 20 microg total RNA and Eberwine amplification of 1 microg total RNA. Starting from as little as 5 ng of total RNA, both methods yielded results in concordance with the Eberwine method.
已经开发出几种用于RNA扩增的方法，使得利用cDNA微阵列分析总RNA量有限的样本成为可能。应用最为广泛的扩增方案是埃伯温法，该方法通过体外转录（IVT）以线性方式扩增RNA。然而，当起始材料限于纳克量的总RNA时，需要进行多轮扩增，这使得该方法既昂贵又费力。通过PCR进行扩增效果稳定，并且能够扩增极其微量的材料。然而，与IVT相比，PCR的非线性特性可能导致扩增的重复性降低。我们评估了两种将PCR和IVT结合起来用于扩增纳克量总RNA的方法。我们将通过这些方法获得的微阵列结果与通过两种既定方法获得的结果进行了比较：对20微克总RNA进行间接标记以及对1微克总RNA进行埃伯温扩增。从低至5纳克的总RNA开始，两种方法均产生了与埃伯温法一致的结果。
