FAME-analysis.md

2.2 Fatty acid methylester (FAME) analysis

The ricinoleic acid content in the castor bean endosperm was determined using gas chromatography-mass spectrometry (GC- MS)-based fatty acid methyl ester (FAME) method (Hielscher et al., 2017). Endosperm tissue was dissected from dry seeds, 24h- imbibed seeds and 1- to 6-day old dark-grown seedlings by removing the embryo and roots with the blunt end of a scalpel blade, weighed and shock frozen in liquid nitrogen. FAME analyses were generated by acid catalyzed methyl-ester formation using methanolic HCl as described by Hielscher et al. (2017). 20 mg endosperm was incubated at 90°C for one hour in 1 ml 3 N methanolic-HCl containing heptadecanoic acid (C17:0) as internal standard. The subsequent extraction of fatty acids from the sample was performed with 1 ml n-hexane and 1 ml 1% (w/v) sodium chloride. Samples were centrifuged at 2,000 g for five minutes. The resulting upper hexane phase was transferred into GC vials. The FAME extracts (1:100 diluted) were analyzed by GC Agilent 7890A gas chromatograph coupled to Waters GCT-TOF Premier mass spectrometer equipped with a Gerstel MPS2XL auto sampler. Quantification and identification of the detected fatty acids was carried out with QuantLynx and MassLynx, respectively. Peaks were integrated and the resulting areas used for determination of relative changes in the abundance of the castor oil-specific ricinoleic acid. As the Ricinus endosperm started to gain fresh weight during seedling development due to water uptake, the fatty acid quantities were normalized to the fresh weight of an average endosperm. All samples were analyzed in three biological replicates.

• Hielscher, B., Charton, L., Mettler-Altmann, T., and Linka, N. (2017). Analysis of peroxisomal b-oxidation during storage oil mobilization in Arabidopsis thaliana Seedlings. Methods Mol. Biol. 1595, 291–304. doi: 10.1007/978-1-4939-6937-1_27