For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative CT (DeltaDeltaCT) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression's CT Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.

Abstract-A human renin/prorenin receptor (RER) has recently been cloned. To gain insight into the molecular function of the RER, we studied its signal transduction mechanisms. Initially, we found a ubiquitous and intracellular expression pattern of the human RER. Consistently, we observed several transcriptional start sites and a high promoter activity of the human RER. We could identify the transcription factor promyelocytic zinc finger (PLZF) protein as a direct protein interaction partner of the C-terminal domain of the RER by yeast 2-hybrid screening and coimmunoprecipitation. Coimmunoprecipitation experiments also indicated homodimerization of the RER. On activation of the RER by renin, PLZF is translocated into the nucleus and represses transcription of the RER itself, thereby creating a very short negative feedback loop, but activates transcription of the p85␣ subunit of the phosphatidylinositol-3 kinase (PI3K-p85␣). Small interfering RNA against the RER abolished these effects. A PLZF cis-element in the RER promoter was identified by site-directed mutagenesis and electrophoretic mobility-shift assay. Renin stimulation caused a 6-fold recruitment of PLZF to this promoter region as shown by chromatin immunoprecipitation. Moreover, renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. These effects were partly abolished by PI3K inhibition and completely abrogated by small interfering RNA against PLZF. Finally, experiments in PLZF knockout mice confirmed the role of PLZF as an upstream regulator of RER and PI3K-p85␣. Our data demonstrate the existence of a novel signal transduction pathway involving the ligand renin, RER, and the transcription factor PLZF, which is of physiological and putative pathophysiological relevance. Key Words: renin receptor Ⅲ PLZF Ⅲ ChIP Ⅲ signal transduction R enin and prorenin are classically thought of as (pro)enzymes of the renin/angiotensin system (RAS), but recent evidence suggests that they also act as hormones because of their ability to bind cellular targets. 1 In 2002 a human renin/prorenin receptor (RER) has been cloned, which consists of 350 amino acids with a single transmembrane domain and specifically binds prorenin and renin. Interestingly, this receptor exerts a dual molecular function 2,3 (1) Binding of renin to its receptor increases the catalytic activity of renin approximately 4-to 5-fold. Furthermore, prorenin, which does not exhibit significant ability to generate angiotensin I in solution, gains enzymatic activity comparable to renin by binding to the RER, ie, the receptor is able to unmask the catalytic activity of prorenin. (2) The RER is also able to induce a signal transduction cascade on ligand binding. Binding of renin and also prorenin causes phosphorylation of the receptor and activation of the mitogen-activated protein kinases ERK 1 and 2 (extracellular signal-regulated kinases 1 and 2), whereas intracellular calcium or cAMP levels are not altered. Remarkably, even deglycosylated renin is able t...

Abstract-Angiotensin II type 2 (AT 2 ) receptors can be regarded as an endogenous repair system, because the AT 2 receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT 2 receptor agonist, compound 21 (C21; dissociation constant for AT 2 receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: Ͼ10 000 nM). This study tested AT 2 receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor B. C21 dose-dependently (1 nM to 1 mol/L) reduced tumor necrosis factor-␣-induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT 2 receptor specificity was controlled for by inhibition with the AT 2 receptor antagonist PD123319 and by the absence of effects in AT 2 receptor-deficient cells. AT 2 receptor-coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor B, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-␣ in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT 2 receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT 2 receptor agonist C21. These data suggest that pharmacological AT 2 receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor B. (Hypertension. 2010;55:924-931.)