Table 1. Primers used for amplifying the Elovl2, Elovl5 and Fads2 ORFs, including the GenBank accession number of the sequence used for primer design. Role of Elongase Reactions in LCPUFA Synthesis Accession number NM_001109118 NM_134382 NM_031344 Gene Elovl2 Elovl5 Fads2 Primer Elovl2-F Elovl2-R Elovl5-F Elovl5-R Fads2-F Fads2-R Sequence 59R39 GCGGAATTCTTGGACAACATGTTTGGACCA GACTGCGGCCGCCGCTTCACCTCATTGCACCTTCTTG CCCGGATCCAAAATGGAACATTTCGATGCG CCGCTCGAGTCAATCCTTCCGGCTGCTTCC GCGGAATTCACAGGCAGCATGGGGAAGGGA GACTGCGGCCGCTCTGCTGCTTCATTTGTGGAG Restriction enzyme sites are indicated by italics and the start and stop codons are underlined. doi:10.1371/journal.pone.0029662.t001 Recombinant yeast expressing Elovl2 or Elovl5 were supple- mented with one or more of the following PUFA substrates: 18:4n- 3 (SDA), 18:3n-6 (c-linolenic acid; GLA), 20:5n-3 (EPA), 20:4n-6 (arachidonic acid; AA), 22:5n-3 (DPA) or 22:4n-6. Recombinant yeast expressing Fads2 were supplemented with one or more of the following putative D6-desaturase substrates: 18:3n-3 (ALA), 18:2n-6 (linoleic acid; LA) or 24:5n-3; D5- desaturase substrates: 20:4n-3 or 20:3n-6; D8-desaturase sub- strates: 20:3n-3 or 20:2n-6. PUFA substrates were prepared in ethanol. The volume of ethanol in each incubation was kept constant. Cells were harvested for analysis after 24 h of fatty acid supplementation at 30uC. The cells were pelleted by centrifugation at 1500 g for 5 min and the cell pellets were washed with 0.85% saline. The wet weights of the cell pellets were recorded. Data are expressed as the means 6 SD of triplicate incubations for each treatment. Elovl2 and Elovl5 expression levels Dark Agouti rats consumed a fat free AIN-93G rodent diet (Glen Forrest Stockfeeders, Glen Forrest, Western Australia) blended with flaxseed oil to obtain 1%en ALA and 2.1%en LA, to a level of 5% (w/w) fat. The experiment was approved by the Animal Ethics Committee of the Institute of Medical and Veterinary Science (125/06) and was performed in accordance with the National Health and Medical Research Council Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Total RNA was extracted from the liver or heart of the rats with the RNeasy kit (Qiagen). The quantity and quality of RNA was determined by measuring the absorbance at 260 and 280 nm (NanoDrop Technologies Inc., DE, USA) and agarose gel electrophoresis. Total RNA (0.5 mg) was reverse transcribed into cDNA using the QuantiTectH Reverse Tran- scription kit (Qiagen). Quantitative reverse transcription-polymer- ase chain reaction (qRT-PCR) was performed using a Rotor-Gene 3000 (Corbett Research) with the QuantiFastTM SYBRH Green PCR kit (Qiagen). Each reaction contained 10 ng of cDNA, 5 ml References 1. Huang YS, Smith RS, Redden PR, Cantrill RC, Horrobin DF (1991) Modification of liver fatty acid metabolism in mice by n-3 and n-6 delta 6- desaturase substrates and products. Biochim Biophys Acta 1082: 319–327. 2. Yamazaki K, Fujikawa M, Hamazaki T, Yano S, Shono T (1992) Comparison of the conversion rates of alpha-linolenic acid (18:3(n - 3)) and stearidonic acid (18:4(n - 3)) to longer polyunsaturated fatty acids in rats. Biochim Biophys Acta 1123: 18–26. 3. James MJ, Ursin VM, Cleland LG (2003) Metabolism of stearidonic acid in human subjects: comparison with the metabolism of other n-3 fatty acids. Am J Clin Nutr 77: 1140–1145. 4. Leonard AE, Bobik EG, Dorado J, Kroeger PE, Chuang LT, et al. (2000) Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids. Biochem J 350: 765–770. QuantiFastTM SYBRH Green PCR master mix and 1 ml of QuantiTectH Primer Assay (Qiagen), in a total volume of 10 ml. The QuantiTectH Primer Assays used were Elovl2 (Rn_E- lovl2_2_SG, QT01683899) and Elovl5 (Rn_Elovl5_1_SG, QT00178059). Elovl2 or Elovl5 PCR products were used to make 10-fold serial dilutions from 106 copies down to 10 copies. The PCR product standard curve equations were used to calculate the absolute copy number of Elovl2 and Elovl5 in rat liver and heart. The cycling conditions were as follows: PCR initial activation step at 95uC for 5 min, 2 step cycling with 35 cycles of denaturation at 95uC for 10 s and annealing/extension at 60uC for 30 s. Each amplification was performed in duplicate. Fatty acid analysis Total lipid was extracted from yeast cells and analysed by gas chromatography as previously described [23]. The identities of 20:4n-3 and 22:4n-3 were previously confirmed by gas chroma- tography–mass spectrometry [23]. The internal free fatty acid standard 17:0 was used to quantify the amount of each endogenous fatty acid. The proportion of substrate fatty acid converted to fatty acid product(s) was calculated as [product(s)/ (product(s) + substrate)]6100. Statistical analysis One-way ANOVA with Tukey’s post-hoc test was performed using Graphpad Prism version 5.03 for Windows (Graphpad Software, San Diego, CA, USA). Statistical significance was set at P,0.05. Author Contributions Conceived and designed the experiments: MG RG MJ. Performed the experiments: MG RC-J RG. Analyzed the data: MG MJ. Contributed reagents/materials/analysis tools: RG LC MJ. Wrote the paper: MG MJ RG LC. 5. Inagaki K, Aki T, Fukuda Y, Kawamoto S, Shigeta S, et al. (2002) Identification and expression of a rat fatty acid elongase involved in the biosynthesis of C18 fatty acids. Biosci Biotechnol Biochem 66: 613–621. 6. Leonard AE, Kelder B, Bobik EG, Chuang LT, Lewis CJ, et al. (2002) Identification and expression of mammalian long-chain PUFA elongation enzymes. Lipids 37: 733–740. 7. Blank C, Neumann MA, Makrides M, Gibson RA (2002) Optimizing DHA levels in piglets by lowering the linoleic acid to alpha-linolenic acid ratio. J Lipid Res 43: 1537–1543. 8. Cleland LG, Gibson RA, Pedler J, James MJ (2005) Paradoxical effect of n-3- containing vegetable oils on long-chain n-3 fatty acids in rat heart. Lipids 40: 995–998. PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29662