Evaluation of TransPlex Whole Transcriptome Amplification method for microarray gene expression profiling

The increased use of microarray expression profiling to study both the molecular biology of cancer and the cellular physiology of difficult-to-isolate cell types has led to a growing need for methods that allow the use of limiting quantities of RNA. This limitation has prompted the development of amplification methods that produce the quantities of RNA required for microarray analysis. Efforts have become increasingly focused upon developing a protocol that minimizes amplification bias, provides versatility, and reduces technical complexity. We evaluated the new protocol Transplex™ Whole Transcriptome Amplification (WTA) produced by Rubicon Genomics. The kit was tested on Human Reference RNA (Stratagene) and on RNA extracted from a renal tumor cell line. Reproducibility, sensitivity and reliability in calling differentially expressed genes were evaluated by both Real-Time PCR and GeneChip® technology (Affymetrix). We tested reproducibility by comparing the expression profiles provided by U133 Plus 2.0 GeneChips® for 3 independently amplified RNA samples: a high degree of correlation between the replicates was obtained. Sensitivity was evaluated by performing WTA on serial dilutions of RNA (from 100 ng to 5 ng). Finally, we evaluated the performance of RNA amplified against identical hybridizations from non amplified RNA; the amplification bias introduced by this method was small. We can conclude that the WTA method is a robust tool with the potential for application in a variety of research settings, in particular when very small amounts of starting template RNA are available. This work was supported by MIUR-FIRB grants n°RBNE01HCKF and n°RBNE01TZZ8