Although successful in significantly reducing RNA requirements, IVT-based amplification procedures have several notable shortcomings. With respect to RNA amplification strategies, T7 polymerase in vitro transcription (IVT) is currently the most commonly used and well documented amplification protocol ( 6). Furthermore, even with this improvement, the amounts of RNA required are still often unobtainable. Various approaches to signal amplification have been described and in some cases have brought down the requirement for RNA by as much as 10-fold ( 1– 5), but these protocols are problematic and expensive to perform. In order to circumvent this issue, a significant amount of effort has been focused on approaches to reduce the RNA requirement per hybridisation through the use of either signal or RNA amplification technologies. In circumstances where the sample available is limited, such as in the case of clinically derived material or the need to focus the analysis on specific cell populations, there is often too little RNA available to perform the analysis using conventional labelling strategies. The need for replicate hybridisations increases the demand for RNA further still. Conventional target labelling protocols however, need relatively large amounts (>20 µg) of input total RNA per hybridisation. Microarray technology is delivering profound insights into numerous biological processes. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identification of differentially expressed genes, but it is also faster and less labour intensive than other amplification protocols. We have also validated the fidelity of amplification and show that the amplified material faithfully represents the starting mRNA population. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identification of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. TS-PCR identifies up to 80% of the differentially expressed genes identified by direct labelling using 30-fold less input RNA for the amplification, with the equivalent of 1000-fold less starting material being used for each hybridisation. Here we explore the cycle-dependent amplification characteristics of Template-Switching PCR and validate its use for microarray target labelling. Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small.
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