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Clinical Chemistry 52: 1161-1167, 2006. First published April 20, 2006; 10.1373/clinchem.2005.062406
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(Clinical Chemistry. 2006;52:1161-1167.)
© 2006 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments

Jochen Wilhelm1,a, Jai Prakash Muyal1, Johannes Best1, Grazyna Kwapiszewska1, Maria Magdalena Stein1, Werner Seeger2, Rainer Maria Bohle1 and Ludger Fink1

Departments of1 Pathology and 2 Internal Medicine, Justus-Liebig-University Giessen, Giessen, Germany.

aAddress correspondence to this author at: Department of Pathology Justus-Liebig-University Giessen, Langhansstrasse 10, 35392 Giessen, Germany. Fax 49-641-9941164; e-mail jochen.wilhelm{at}patho.med.uni-giessen.de.

Background: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification.

Methods: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMARTTM for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases.

Results: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases.

Conclusions: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.






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Copyright © 2006 by the American Association for Clinical Chemistry.