Molecular beacons can assess changes in expression and 3'-polyadenylation of human eNOS mRNA.
The endothelium plays an essential role in maintaining vascular homeostasis, and it fulfills this role by modulating intracellular signaling and gene expression in response to chemical and mechanical stimuli. Assessing changes in endothelial gene expression is essential to understanding how physiological and pathophysiological processes modulate vascular homeostasis. Here we describe the use of molecular beacons to rapidly and quantitatively assess expression and 3'-polyadenylation of a gene that is important for vascular homeostasis, endothelial nitric oxide synthase (eNOS). Single- and dual-fluorescence resonance energy transfer (FRET) molecular beacon hybridization assays were developed to measure changes in mRNA levels and 3'-polyadenylation, respectively, in primary human endothelial cell cultures subjected to laminar shear stress or statin treatment. Optimized beacon hybridization assays took approximately 15 min to perform, and eNOS mRNA levels were validated by quantitative real-time RT-PCR. Competitive inhibition assays and posttranscriptional silencing of eNOS expression were used to verify the specificity of molecular beacon fluorescence. Finally, the dual-FRET method was used to assess eNOS polyadenylation in tissues isolated from mice subjected to exercise training. These data demonstrate that molecular beacons can be used to rapidly and efficiently measure endothelial gene expression and 3'-polyadenylation. This approach could easily be adapted for studies of other endothelial genes and has promise for applications in live endothelial cells.
Jones
Rachel
R
Div. of Cardiology, Emory Univ. School of Medicine, Atlanta, GA 30322, USA.
Baker
Meredith B
MB
Weber
Martina
M
Harrison
David G
DG
Bao
Gang
G
Searles
Charles D
CD
D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D008168:Lung / Q000201:enzymology; D051379:Mice; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D005082:Physical Exertion; D026723:Polyadenylation; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D015203:Reproducibility of Results; D013314:Stress, Mechanical
D005456:Fluorescent Dyes; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C012258:mevastatin; D008148:Lovastatin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse