sex ratio, sampling time and RNA quality 9. Lipid extraction and fatty acid analysis One gram of white muscle was dissected from 15 selleckbio fish per dietary treatment and immediately frozen in liquid nitrogen. Total lipids were extracted according Inhibitors,Modulators,Libraries to the method of Folch et al. by Accelerated Solvent Extraction 200 with dichloromethane methanol containing 0. 01% butylatedhydroxuto luene as antioxidant. Lipids were extracted at 100 bars, 100 C, with a 5 min precallingphase, 2 min static phase, and 60% flush for 60 sec. The separation of neutral and polar lipids was performed according to the procedure described by Juaneda and Roquelin. The total lipid extracts were fractio nated on silica cartridges, neutral lipids were eluted with chloroform and polar lipids with methanol. Fatty acid methyl esters trans esterification.
FAME were quantified by gas liquid chro matography with a BPX70 column of 30 m length Inhibitors,Modulators,Libraries and 0. 22 mm I. D. Hydrogen was used as carrier Inhibitors,Modulators,Libraries gas and temperature programming was from 50 C to 180 C at 20 C min and then to 220 C at 3 C min. Individual methyl esters were identified by com parison with known standards. The fatty acid analysis was performed on one sample per fish. RNA extraction and real time quantitative PCR analysis Total mRNA of liver was extracted using Trizol reagent and quantified by measuring absor bance at 260 nm in a spectrophotometer. The reverse transcription was per formed using the QuantiTect Reverse Transcription kit, including a genomic DNA elimination reac tion.
Reactions were carried out in a volume of 20 ul, containing 1 ug of total RNA, 1 unit of Quantiscript Reverse Transcriptase, 4 ul of Quantiscript RT buffer and 1 uM primer mix. Seven genes involved in metabolic and or immune pathways of interest, whose the oligonucleotides were spotted on the chip, were Inhibitors,Modulators,Libraries analysed by real time PCR in order to validate the gene expression patterns obtained through the microarray approach. The relative mRNA levels were automatically normalized with housekeeping elongation factor 1 gene expression and measured by Bio Rad IQ5 software using Ct method, Gene Normalized Expression in sample 1 with Relative Quantity in sample 1 for a gene i E Ct Ct Ef1 was chosen to provide an internal control for real time PCR, since contrary to 18S rRNA and actin initially tested, we did not observe any significant difference between Ct values for Ef1 between the dietary groups.
Its stability was also assessed by a low coefficient of varia tion over all the samples. Specific primers were designed from Eur opean sea bass sequences of fads2, fabp7, AV-951 hmgcr, angptl3, cxcl10, gck, lpl and ef1. All primers used for real time quantitative PCR analysis were defined with the Primer3 software primer3 in order to respect an annealing tem perature of 60 C. All PCR reactions were performed with an efficiency of 100%. The PCR reactions were carried out in an I cycler with an optical module, in a final volume of 15 Trichostatin A ul containing 7. 5 ul SYBR Green Supermix, 0. 5 ul of e