Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways

摘要: The current interest in expression of groups functionally related genes creates a demand for novel experimental tools. We describe multiplex ligation-dependent amplification procedure (RT-MLPA), which accurately quantifies up to 45 transcripts one-tube assay. output, set fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. highly sensitive reproducible over 100-fold range input RNA, with excellent compatibility RT-PCR microarrays. targeted two comprehensive sets human genes: 35 apoptosis regulators 30 involved inflammation. Both probe assessed specific changes gene relevant model systems. Stimulation lymphocytes various Toll-like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated cytostatic drugs showed as primary response strong up-regulation the apoptogenic p53-inducible PUMA transcript. Suppression by RNAi validated that indeed Puma was responsible induction. Thus, RT-MLPA enables transcription patterns be quickly pinpointed guide further experiments. This can an advantage compared hypothesis-free whole genome screens where large numbers differentially expressed obscure functional interpretation.