The ProbeLibrary™ - Expression profiling 99% of all human genes using only 90 dual-labeled real-time PCR Probes

While quantitative real-time RT-PCR is in principle a simple technique, the assay design remains fairly complex and designed assays often perform inadequately with respect to specificity, sensitivity, and reproducibility (1,2). The time spent on assay design, optimization, and validation often becomes a bottleneck in the implementation of new assays for large-scale expression profiling. Commercially available pre-validated real-time RT-PCR assays simplify the assay development process, but the time of delivery sometimes causes delays in experimental progress. Furthermore, prevalidated probe-based assays lack flexibility, due to the fact that these assays target a specific site in a given transcript. Consequently, quantification of another transcript or splice variant requires a different assay. Here we describe a novel, highly flexible concept for quantitative real-time RT-PCR, based on the development of a ProbeLibraryTM of 90 prevalidated real-time PCR detection probes, and a new web-based assay design software, enabling fast and easy design of optimal real-time PCR assays for gene expression analysis. By combining individual ProbeLibraryTM probes and target-specific PCR primers selected using the Primer3 software (3), the Assay Design Center software is able to design more than 644,000 different assays in the human transcriptome or target 98% of all human transcripts (Figure 1, Table 1). PRINCIPLES OF THE TECHNOLOGY