Fatty Acids Stability Effect on Rns

DSF was performed to assess the effect of fatty acids on Rns stability^{40 (link)}. First stocks of fatty acids at 100× of final concentration were made by adding octanoic, decanoic, and palmitic acid to methanol. The decanoic acid was serially diluted by ½, in methanol, to obtain the concentrations for the dose response. 1 μl of the appropriate fatty acid was added to 99 μl of Rns, at a concentration of ~ 0.7 mg/ml, and incubated at room temperature for 1 h. Then 18 μl of the mixture was added to a PCR plate in triplicate. Sypro Orange dye (Life Technologies), diluted in buffer, was added to the PCR plate for a final concentration 5x, and a total reaction volume of 20 μl. For each condition a buffer only control was also performed in triplicate. The melting curves were generated using a StepOne + RT PCR machine (Life Technologies) with a gradient from 25 to 95 °C utilizing 1 °C steps. The normalized melt data was exported into STATA for analysis as described^{41 (link)}.