Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments
1 Department of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg 69120, Germany
2 Department of Gynecology and Obstetrics, Städtische Kliniken Bielefeld, Bielefeld 33604, Germany
3 Institute of Molecular Biology and Cell Culture Technology, Mannheim University of Applied Sciences, Mannheim 68163, Germany
BMC Genomics 2004, 5:29 doi:10.1186/1471-2164-5-29Published: 30 April 2004
The requirement of a large amount of high-quality RNA is a major limiting factor for microarray experiments using biopsies. An average microarray experiment requires 10–100 μg of RNA. However, due to their small size, most biopsies do not yield this amount. Several different approaches for RNA amplification in vitro have been described and applied for microarray studies. In most of these, systematic analyses of the potential bias introduced by the enzymatic modifications are lacking.
We examined the sources of error introduced by the T7 RNA polymerase based RNA amplification method through hybridisation studies on microarrays and performed statistical analysis of the parameters that need to be evaluated prior to routine laboratory use. The results demonstrate that amplification of the RNA has no systematic influence on the outcome of the microarray experiment. Although variations in differential expression between amplified and total RNA hybridisations can be observed, RNA amplification is reproducible, and there is no evidence that it introduces a large systematic bias.
Our results underline the utility of the T7 based RNA amplification for use in microarray experiments provided that all samples under study are equally treated.