Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study

  • Authors:
    • Giovanna Albertin
    • Gianni Carraro
    • Gastone G. Nussdorfer
  • View Affiliations

  • Published online on: April 1, 2005     https://doi.org/10.3892/ijmm.15.4.579
  • Pages: 579-583
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Abstract

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where endogenous AM system had been suppressed. Hence, we developed a protocol to silence the human AM gene by transfection with short interfering RNAs (siRNAs). Eight possible AM-siRNA sequences were designed: six siRNAs were synthesized in our laboratory and two were provided by Ambion. As positive control the suppression of the glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene was tested using the Ambion Silencer GADPH siRNA kit. Cultured human embryonal kidney cell line HEK-293 and human umbilical vein endothelial cells (HUVECs) were transfected using either the Qiagen or the Ambion transfection reagent, and transfection visualization, carried out using Cy3-labeled siRNA and examining red fluorescence within the cells, showed that the former reagent was the most efficient. AM-gene silencing was determined in HUVECs by measuring AM mRNA levels in transfected and control cells by real-time polymerase chain reaction. Only Ambion siRNAs were effective, and the best AM-gene silencing (about 80%) was observed 48 or 72 h after transfection with 3 or 6 µg of siRNAs. The conclusion is drawn that siRNA technology can be useful in the investigations on AM functions, but that the complete suppression of the AM-gene transcription is very difficult to obtain.

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April 2005
Volume 15 Issue 4

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Spandidos Publications style
Albertin G, Carraro G and Nussdorfer GG: Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study. Int J Mol Med 15: 579-583, 2005.
APA
Albertin, G., Carraro, G., & Nussdorfer, G.G. (2005). Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study. International Journal of Molecular Medicine, 15, 579-583. https://doi.org/10.3892/ijmm.15.4.579
MLA
Albertin, G., Carraro, G., Nussdorfer, G. G."Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study". International Journal of Molecular Medicine 15.4 (2005): 579-583.
Chicago
Albertin, G., Carraro, G., Nussdorfer, G. G."Human adrenomedullin gene silencing by short interfering RNAs: A preliminary study". International Journal of Molecular Medicine 15, no. 4 (2005): 579-583. https://doi.org/10.3892/ijmm.15.4.579