Purchase this article with an account.
D. L. Harris, N. C. Joyce; p21WAF1/cip1 siRNA Electroporation of Primary Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1805.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To test p21WAF1/cip1 siRNA electroporation in primary human corneal endothelial cells (HCEC) from young (< 30yo) and older donors (>50yo).
Confluent cultures of HCEC were trypsinzed and electroporated using a Nucleofector II Device from Amaxa, Inc. (Gaithersburg, MD). Optimal conditions were determined using the kit for primary mammalian endothelial cells by electroporating with a pmax GFP plasmid or a Cy3-labeled non-silencing siRNA at 0.5, 1, 2, and 3 ug and viewing efficiency with a fluorescence microscope. Further optimization included testing of 3 different p21WAF1/cip1 siRNAs (Ambion, Austin, TX). Controls included cells not electroporated or electroporated with non-silencing siRNA. To determine the most efficient down-regulation of the corresponding protein, cells were electroporated, seeded in dishes, maintained for 72 hrs in 8% FBS, followed by western blot analysis with p21WAF1/cip1, p16INK4a and beta-actin antibodies.
The program T-023 using 1 ug of siRNA was the most efficient for siRNA electroporation in HCEC. All three p21WAF1/cip1 siRNAs significantly reduced p21WAF1/cip1 protein expression using beta-actin for normalization. This treatment did not affect expression of p16INK4a. p21WAF1/cip1 protein levels were significantly reduced in HCEC from older donors compared with young donors.
This PDF is available to Subscribers Only