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■ Abstract Real-time polymerase chain reaction (PCR) is commonly used for a sensitive and specific quantification of messenger RNA (mRNA). The levels of mRNA are frequently compared between two or more experimental groups. However, such comparisons require normalization procedures, and reference genes are frequently used for this purpose. We discuss pitfalls in normalization and specifically in the choice of reference genes. Reference genes, which prove suitable for some experimental conditions, are not necessarily similarly appropriate for others. Therefore, a proper validation of the suitability of a given reference gene or sets thereof is required for each experimental setting. Several computer programmes are available to aid such validation.

Objective-In addition to their role in programmed cell death, cell survival, and cell growth, sphingolipid metabolites such as ceramide, sphingosine, and sphingosine-1-phosphate have vasoactive properties. Besides their occurrence in blood, they can also be formed locally in the vascular wall itself in response to external stimuli. This study was performed to investigate whether vasoactive compounds modulate sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses. Methods and Results-In isolated rat carotid arteries, the contractile responses to angiotensin II are enhanced by the sphingosine kinase inhibitor dimethylsphingosine. Endothelium removal or NO synthase inhibition by N -nitro-Larginine results in a similar enhancement. Angiotensin II concentration-dependently induces NO production in an endothelial cell line, which can be diminished by dimethylsphingosine. Using immunoblotting and intracellular calcium measurements, we demonstrate that this sphingosine kinase-dependent endothelial NO synthase activation is mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways. Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to form ceramide. 1,2 The sequential action of ceramidase and sphingosine kinase converts ceramide to sphingosine and sphingosine-1-phosphate (S1P), and ceramide synthase and S1P phosphatase can reverse this process to form ceramide from S1P. 3,4 The sphingomyelin metabolites ceramide, sphingosine, and S1P are biologically active mediators that play important roles in cellular homeostasis. In this regard, ceramide and sphingosine on the one hand and S1P on the other hand frequently have opposite biological effects. For example, ceramide and sphingosine are generally involved in apoptotic responses to various stress stimuli and in growth arrest, 5,6 whereas S1P is implicated in mitogenesis, differentiation, and migration. 7,8 This homeostatic system is frequently referred to as the ceramide/S1P rheostat. 9 It can be hypothesized that this rheostat also plays a role in vascular contraction and relaxation because S1P, sphingosine, and ceramide are potentially counteracting, vasoactive compounds. 10,11 The molecular basis of ceramide effects has not been explored fully but is believed to involve stress-activated protein kinases, protein phosphatases such as protein phosphatases 1 and 2, guanylyl cyclase, and charybdotoxinsensitive K ϩ channels. 11,12 The molecular basis of S1P effects has been characterized in more detail. S1P can act on specific G protein-coupled receptors, of which 5 subtypes have been identified thus far, termed S1P 1-5 . These receptors couple to intracellular second messenger systems including intracellular Ca 2ϩ , adenylyl cyclase, phospholipase C, phosphatidylinositol 3 (PI3)-kinase, protein kinase Akt, mitogen-activated protein kinases, and Rho-and Ras-dependent pathways. 13 The cardiovascular system primarily expresses the receptor subtypes S1P 1-3 , and within the vasculature they are express...