Transcript profiling methods

Optical gene chips dense arrays of oligonucleotides have been successfully applied to detect transcriptional profiling and SNP discovery, where massively parallel analysis is required. However, the fluorescence-based readout of these chips involves not only highly precise and expensive instrumentation but also sophisticated numerical algorithms to interpret the data, and therefore these methods have been commonly limited to use in research laboratories. In this way, thin-film arrays of 14, 20, 25, 48 and 64 electrodes have already been fabricated [12,15,39,40,44,48], using lithographic techniques. Readout systems for these arrays based on electrical detection have also been developed. 

This chapter will discuss various experimental approaches used to select the relevant species for conduct of toxicology studies for biopharmaceuticals, as well as highlight advances made in scientific approaches and technologies to facilitate this process. Methods discussed include the traditional immunohisto-chemistry and tissue cross-reactivity studies, flow cytometry, protein sequencing, and functional in vitro assays, as well as newer approaches such as utilization of microarray databases for genomic mRNA expression data and use of transcript profiling studies as an adjunct to functional assays, to understand similarity in pharmacological responsiveness between animals and humans. 

Because of the preceding properties, our profile procedure appears to produce highly sensitive and specific common pattern representations from limited numbers of defining sequences compared with other current methods (Figs. 5 and 7). This was shown by the construction of such profiles from more than 50 completely unrelated functional families. In more than 90% of the families, the sensitivity and specificity are more than 98%. This is also supported by the repeated sampling study of the complex bacterial transcription initiation factors. Finally, these methods allow for the localized recognition of entire domains within multidomain structures, as seen in Fig. 6. 

Unlike other methods currently employed for quantitative transcript measurements, including cDNA microarrays and real-time RT-PCR, competitive RT-PCR is amenable to quality control, which is critical for clinical diagnostic and pharmaceutical industry applications. Furthermore, microarray approaches are limited to generating snap-shot like profiles, but they do not control for differences in hybridization efficiencies of different gene probes with their corresponding cDNAs. That is, cross comparisons are relative and not absolute. Real-time PCR has gained acceptance recently largely due to the reduced cost associated... 

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