Ontology type: schema:ScholarlyArticle
2007-10-31
AUTHORSMartin Lauss, Klemens Vierlinger, Andreas Weinhaeusel, Sandra Szameit, Klaus Kaserer, Christa Noehammer
ABSTRACTAvailable ribonucleic acid (RNA) amplification methods are extensively tested for reproducibility, but only a few studies additionally deal with potential amplification bias. On targeted arrays, we evaluated three amplification protocols, which are less time consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template-switching polymerase chain reaction (PCR), Ribo-single primer isothermal amplification and a random primer-based PCR. Additionally, a more sensitive labelling method, Dendrimer labelling, was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. From our results, we conclude that RNA amplification with template-switching PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with template-switching PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. Template-switching PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number of 4.3. In conclusion, template-switching PCR amplification promises to help micro-array expression profiling of limited amounts of human samples on its way to a clinical routine. More... »
PAGES1019-1029
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DOIhttp://dx.doi.org/10.1007/s00428-007-0522-4
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