Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways.
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.
Eldering
Eric
E
Department of Experimental Immunology, Academical Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. e.eldering@amc.uva.nl
Spek
C Arnold
CA
Aberson
Hella L
HL
Grummels
Annette
A
Derks
Ingrid A
IA
de Vos
Alex F
AF
McElgunn
Cathal J
CJ
Schouten
Jan P
JP
D017209:Apoptosis / Q000235:genetics; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation / Q000235:genetics; D019169:Jurkat Cells; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D012516:Osteosarcoma / Q000235:genetics / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D012984:Software; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics
D015342:DNA Probes; D012333:RNA, Messenger