Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes.

Posttranscriptional control of renin synthesis: identification of proteins interacting with renin mRNA 3'-untranslated region.

Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3'-untranslated region (3'-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed.

Suppression of 15-lipoxygenase synthesis by hnRNP E1 is dependent on repetitive nature of LOX mRNA 3'-UTR control element DICE.

Cytidine-rich 15-lipoxygenase differentiation control element (15-LOX DICE) is a multifunctional cis-element found in the 3'-UTR of numerous eukaryotic mRNAs. It binds KH domain proteins of the type hnRNP E and K, thus mediating mRNA stabilization and translational control. Translational silencing is caused by formation of a simple binary complex between DICE and recombinant hnRNP E1 (E1).