Designing a unique reporter based microarray sensor platform for monitoring cell signalling.

Abstract

PurposeBiological processes in mammalian cells are driven by gene expression that is in turn regulated by transcription factors (TFs). The latter are controlled by a series of complex network of biochemical pathways that represent the core for cell signalling. There has been little success in monitoring cell signalling using traditional methods such as proteomics. In this report we demonstrate the possibility of deconvoluting such complexity using a multiplexed library of plasmids carrying a TF binding site upstream of a unique reporter sequence (UR). A microarray sensor platform was then used to monitor dynamic changes in cells by measuring UR expression levels.MethodsHEK293 cells, treated with various drugs and inducers, were transfected with the multiplexed plasmid library. mRNA was then purified and reverse transcribed to produce first strand cDNA. The latter was then amplified by PCR using a universal fluorescently labelled primer. Purified PCR amplicons were then hybridized with antisense oligonucleotide captures at 53°C. Epoxy slides were then washed and quantified using the ScanArray Express Scanner (Perkin Elmer).ResultsThe microarray platform was optimized to detect down to 1 attomol with a maximal degree of specificity and a minimal probe volume of 2μl. This platform was able to detect changes in the TF activity following treatment of cells with dexamethazone, cadmium, forskolin, TPA, cAMP analogues, cGMP analogues and PDEIs. The data was consistent with qPCR and western blot analysis.ConclusionThe microarray platform was shown to be robust, sensitive and reproducible in monitoring dynamic changes in mammalian cells. The system can be easily tailored to include transcription factors of interest to fit with the required purposes.