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P. Russell, E. Walsh, W. Chen, A. Goldwich, E. Tamm; The Effect of Temperature on Gene Silencing by SiRNAs: Implications for Tissues in the Anterior Chamber of the Eye . Invest. Ophthalmol. Vis. Sci. 2006;47(13):831.
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The folding or secondary structure of a specific mRNA has been found to be significantly correlated to the silencing of mRNAs by siRNAs. Since this base pairing is dependent on free energy, the temperature of the cells may influence the effectiveness of a particular siRNA. The aqueous humor of the human eye has been measured to be around 34°C with the lens acting as a thermal barrier in the eye. We wished to investigate the relationship between cell temperature and gene silencing since this might be a consideration with the use of siRNAs in tissues of the anterior chamber.
Human EBNA 293 cells, which had been transfected to express myocilin, were used in these studies. Five different siRNAs that had been predicted to silence myocilin were studied at three different temperatures, 33°C, 35°C and 37°C. The effects of silencing were compared using real–time PCR on RNA extracted from the cells.
Three different patterns of silencing emerged when the silencings were compared with cells grown at 33°C, 35°C, and 37°C. For one of the siRNAs, the silencing was increased at lower temperatures. For two siRNAs, no significant changes in silencing were observed with different temperatures. Two of the siRNAs were significantly influenced by temperature with little if any silencing occurring at the lowest temperature.
The results support the hypothesis that the local structure of an mRNA can influence silencing by siRNAs. Changes in local structure with temperature are possible and these alterations can significantly influence silencing. These data indicate that siRNAs directed to tissues in the anterior chamber of the eye should be checked at temperatures lower than 37°C to determine their effectiveness.
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