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- Title
Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways.
- Authors
Eldering, Eric; Spek, C Arnold; Aberson, Hella L; Grummels, Annette; Derks, Ingrid A; de Vos, Alex F; McElgunn, Cathal J; Schouten, Jan P
- 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.
- Publication
Nucleic acids research, 2003, Vol 31, Issue 23, pe153
- ISSN
1362-4962
- Publication type
Journal Article
- DOI
10.1093/nar/gng153