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

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