Quantification of mRNA for Endothelial NO Synthase in Mouse Blood Vessels by Real-Time Polymerase Chain Reaction

Abstract

Abstract-The mouse is useful in studies of vascular biology because of its well-defined genetics and because the mouse genome can be manipulated. However, because only small amounts of mRNA can be extracted from blood vessels, the quantification of gene expression in individual mice is difficult. Endothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth. In addition, there appear to be sex differences in the production of NO under basal conditions in mouse aortas. The goals of this study were to develop a real-time polymerase chain reaction (PCR) method to quantify eNOS mRNA in blood vessels from mice and to examine eNOS mRNA levels in vessels from male and female mice. Blood vessels were isolated from C57BL/6 mice. Total RNA from individual mice was isolated and reverse-transcribed. The number of molecules of eNOS mRNA (after reverse transcription) was determined against cDNA standards, with 18S rRNA used as a control for RNA input and reverse-transcription efficiency. When expressed as copy numbers per nanogram of total RNA or as the ratio of eNOS to 18S rRNA, eNOS mRNA was lower in the aortas of female mice than in those of male mice at 7 to 9 months of age. In contrast, no difference in eNOS mRNA was found in the aortas of 2-month-old mice. In addition, eNOS mRNA levels were similar in the carotid, cerebral, and coronary arteries. These findings provide the first quantitative measurements of eNOS mRNA by using real-time PCR in the vessels of mice and suggest age-and sex-related differences in the basal levels of eNOS mRNA in mice. In addition, the eNOS region that was used for real-time PCR was amplified and sequenced for monkeys and other species. Key Words: endothelial NO synthase Ⅲ real-time polymerase chain reaction Ⅲ mice Ⅲ aortas Ⅲ sex E ndothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth in large arteries and in the microcirculation. 1,2 In addition to mechanisms that regulate enzyme activity acutely, eNOS is regulated at mRNA and protein levels. 3,4 Steady-state levels of eNOS mRNA are increased by shear stress, 5 estrogen, 6 and exercise, 7 but these levels are decreased during heart failure 8 and possibly other disease states. A sensitive and reproducible method to quantify eNOS expression is of great importance in studies of vascular physiology and pathophysiology.Quantification of relative eNOS mRNA levels has been accomplished previously by using Northern blotting, 5,7,8 RNase protection assay, 6 and competitive reverse transcription (RT)-polymerase chain reaction (PCR). 9 Real-time PCR (after RT) is a new, sensitive, and accurate method for the quantification of mRNA levels 10 and has had limited application in vascular studies. 11 It requires a small amount of starting RNA and uses the initial (linear) rate of PCR (C T ) as the measure of the original amount of cDNA. With at least 5 orders of linear dynamic range, there is no need to analyze dilutions of each sample, and this, coupled with no need for post-PCR pro...

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“…Probes and primers were designed using Beacon Designer 2.0 software (Premier Biosoft International) and generated at Integrated DNA Technologies (Coralville) with the exception of eNOS, which has been published. 22 Thoracic aorta from vehicle-and rosiglitazonetreated R ϩ A ϩ mice were used to isolate membrane and cytosolic protein fractions. Commercially available antibodies for ␤1 subunit of soluble guanylyl cyclase (Cayman Chemical) and eNOS (Santa Cruz) were used to assess protein expression.…”

Vascular effects of a common gene variant of extracellular superoxide dismutase in heart failure

“…Reverse transcription of RNA was performed using random hexamers as primers. The expression levels of three isoforms of endogenous SODs (CuZnSOD, Mn-SOD, and ecSOD) and eNOS were determined by quantitative realtime PCR using the TaqMan method and normalized against 18S rRNA as previously described (6). The primers and probes for SODs and eNOS listed in Table 1.…”

Role of oxidative stress in elevated blood pressure induced by high free fatty acids

“…The sequences of the primers used were described earlier. 29 The sense primer, 5¢-CCTTCCGCTACCAGCCTGA-3¢, and the antisense primer, 5¢-CAGAGATCTTCACTGCATTGGCTA-3¢, were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd (Shanghai, China). The dual-labeled probe, 5¢-6-carboxy fluorescein-CAGG-CATCACCAGGAAGAAGACCTTTAAGGA-6-carboxy tetramethylrhodamine-3¢, was synthesized by PE Biosystems (Foster City, CA, USA).…”