Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples

Medical University of Vienna, Wien, Vienna, Austria
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin (Impact Factor: 2.65). 01/2008; 451(6):1019-29. DOI: 10.1007/s00428-007-0522-4
Source: PubMed


Available 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.

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    • "A major concern is the amplification method and how it affects the ability to maintain the relative amount of the starting RNA, the so called fidelity of amplification [22]. This was evaluated by comparing the expression of eight genes, which included the three endogenous control genes (PPIA, TBP and GAPDH), three transcription factors involved in the late B-cell lymphopoiesis (XBP1, IRF4 and PRDM1), and two genes related to oncogenesis of MM (MGST1 and WHSC1) in six amplified and non-amplified CCLs [see Additional file 4]. "
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