Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Martino, Alessandro Di; Cabiati, Manuela; Campan, M.; Prescimone, Tommaso; Minocci, D.; Caselli, Chiara; Rossi, Anna Maria; Giannessi, Daniela; Ry, Silvia Del

doi:10.1016/j.jbiotec.2011.04.002

Cited by 51 publications

(36 citation statements)

References 30 publications

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“…Total RNA was collected (RNeasy Kit, Qiagen), and cDNA was generated using Oligo d(T) (Invitrogen) and MML-V reverse transcriptase (Promega). PCR was performed using primers specific for GPR107 and the housekeeping gene, HPRT-1 (12). Amplicons were separated on a 1% agarose gel, and imaged using a FujiFilm Luminescent Image Analyzer (LAS-3000).…”

Section: Methodsmentioning

confidence: 99%

Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107

Yosten

Redlinger

Samson

2012

American Journal of Physiology-Regulatory, Integrative and Comp

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Section: Methodsmentioning

confidence: 99%

Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107

Yosten

Redlinger

Samson

2012

American Journal of Physiology-Regulatory, Integrative and Comp

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“…Quantitative real-time PCR (MyIQ, Bio-Rad) was performed with SYBR green (Bio-Rad) (62). Target gene mRNA levels were expressed relative to the housekeeping gene GAPDH as an endogenous control (39).…”

Section: Quantitative Real-time Pcr Analysismentioning

confidence: 99%

Phosphodiesterase 5 inhibition-induced coronary vasodilation is reduced after myocardial infarction

Merkus

Visser

Houweling

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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DJ. Phosphodiesterase 5 inhibition-induced coronary vasodilation is reduced after myocardial infarction. Am J Physiol Heart Circ Physiol 304: H1370 -H1381, 2013. First published March 15, 2013 doi:10.1152/ajpheart.00410.2012The balance between the production and removal of cGMP in coronary vascular smooth muscle is of critical importance in determining coronary vasomotor tone and thus in the regulation of coronary blood flow. cGMP production by soluble guanylyl cyclase is activated by nitric oxide (NO), whereas cGMP breakdown occurs through phosphodiesterase 5 (PDE5). We hypothesized that myocardial infarction (MI) alters the balance between the production and removal of cGMP in the coronary vasculature and thereby alters the control of coronary vasomotor tone. Chronically instrumented swine with and without a 2-wk-old MI were exercised on a treadmill in the absence and presence of the PDE5 inhibitor EMD-360527 (300 g·kg Ϫ1 ·min Ϫ1 iv). Inhibition of PDE5 produced coronary resistance vessel dilation, which was more pronounced at rest than during exercise in normal swine. PDE5 gene expression was markedly reduced in coronary resistance vessels isolated from the remote myocardium of MI swine, which was accompanied by a similarly marked attenuation of coronary vasodilation by PDE5 inhibition in MI swine. The coronary vasoconstriction produced by inhibition of NO synthesis with N -nitro-L-arginine (20 mg/kg iv) was only slightly smaller in swine with MI. Interestingly, inhibition of NO synthesis reduced the vasodilator response to subsequent PDE5 inhibition in normal swine but not in MI swine. Conversely, PDE5 inhibition enhanced the coronary vasoconstriction produced by NO synthesis inhibition in normal swine but not in MI swine, suggesting that downregulation of PDE5 mitigated the loss of NO vasodilator influence. In conclusion, the expression and vasoconstrictor influence of PDE5 are markedly attenuated in coronary resistance vessels in the remote myocardium after MI, which appears to serve as a compensatory mechanism to mitigate the loss of NO vasodilator influence. coronary circulation; myocardial infarction; nitric oxide; phosphodiesterase 5; vasodilation LEFT VENTRICULAR (LV) function critically depends on an adequate oxygenation of the myocardium. Since myocardial O 2 extraction (ME O 2 ) is already high under resting conditions, any substantial increase in myocardial O 2 demand must be met by an increase in O 2 supply and hence an increase in coronary blood flow (CBF) (15,20). This tight matching of CBF to myocardial metabolism is the result of a delicate balance between a myriad of vasomotor signals stemming from cardiomyocytes, autonomic nerve terminals, and the endothelium (11,15,20,56).The endothelium-derived vasodilator nitric oxide (NO) produces coronary vasodilation through the activation of soluble guanylyl cyclase (sGC) and formation of cGMP in vascular smooth muscle (21). cGMP signaling is terminated through breakdown of cGMP by phosphodiesterase 5 (PDE5) and, to a lesser extent, by PDE1 (46). Th...

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“…It was very important to select the appropriate reference genes in the realtime PCR. Thus, a reference gene group including β-actin, TATA box binding protein (TBP) and GAPDH was chosen for both in vivo and in vitro studies according to the relevant references (18,21,(24)(25)(26)(27). In addition, the geometric mean of the expression of the reference genes was used to confirm the robustness of experimental data (28).…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

Serum Amyloid A Directly Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

Dong

Qin

et al. 2011

Mol Med

111

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Although serum amyloid A (SAA) is an excellent marker for coronary artery disease, its direct effect on atherogenesis in vivo is obscure. In this study we investigated the direct effect of SAA on promoting the formation of atherosclerosis in apolipoprotein E-deficient (ApoE -/-) mice. Murine SAA lentivirus was constructed and injected into ApoE -/-mice intravenously. Then, experimental mice were fed a chow diet (5% fat and no added cholesterol) for 14 wks. The aortic atherosclerotic lesion area was larger with than without SAA treatment. With increased SAA levels, the plasma levels of interleukin-6 and tumor necrosis factor-α were significantly increased. Macrophage infiltration in atherosclerotic regions was enhanced with SAA treatment. A migration assay revealed prominent dose-dependent chemotaxis of SAA to macrophages. Furthermore, the expression of monocyte chemotactic protein-1 and vascular cell adhesion molecule-1 (VCAM-1) was upregulated significantly with SAA treatment. SAA-induced VCAM-1 production was detected in human aortic endothelial cells in vitro. Thus, an increase in plasma SAA directly accelerates the progression of atherosclerosis in ApoE -/-mice. SAA is not only a risk marker for atherosclerosis but also an active participant in atherogenesis.

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Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Cited by 51 publications

References 30 publications

Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107

Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107

Phosphodiesterase 5 inhibition-induced coronary vasodilation is reduced after myocardial infarction

Serum Amyloid A Directly Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

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