Gene silencing by siRNAs offers the potential for reducing the expression of mutated genes that cause disease. It has been shown that the folding or secondary structure of a specific mRNA was significantly correlated to the silencing observed. 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 degrees C with the lens acting as a thermal barrier in the eye. The trabecular meshwork, bathed by the aqueous humor, probably has a temperature lower than body temperature under normal conditions. Mutated myocilin, that is associated with primary open angle glaucoma, would appear to be a candidate for silencing by siRNAs. Silencing of the mutated myocilin might prevent additional accumulation of this protein in the rough endoplasmic reticulum. These experiments were undertaken to determine the influence of lowered temperatures on the silencing of myocilin by five siRNAs. Three different patterns of silencing emerged when the silencings were compared with cells grown at 33, 35, and 37 degrees 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. These data indicate that siRNAs directed to tissues in the anterior chamber of the eye should be checked at temperatures lower than 37 degrees C to determine their effectiveness.