One such factor could be starvation or individual differences in the time of the last food intake
One such factor could be starvation or individual differences in the time of the last food intake. levels of lathosterol, 24S-hydroxycholesterol or cholesterol in this organ. The data obtained are discussed in relation to inconsistent effects of n-3 PUFAs on serum lipids in human trials and reported positive effects of fish oil on cognitive function. for 15 min to separate the serum. T-C, HDL cholesterol (HDL-C), LDL-C and triglycerides (TG) in the serum were measured in triplicate using commercial enzymatic kits (Stanbio Laboratory, Boerne, TX). 2.3. Cloning of the hamster cDNAs for CYPs 7A1, 46A1, and 27A1 Total RNA was isolated using the RNAqueous Kit (Ambion, Austin, TX) from the stored frozen samples of the GDC0853 hamster brain cortex, liver and eyeballs that were placed in the RNAlater answer (Ambion, Austin, TX) immediately after the organ excision. The quality of the RNA samples was assessed by the analysis on an RNA Nano chip using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Rabbit Polyclonal to MPHOSPH9 Clara, CA). Synthesis of cDNA was performed using 1 g of total RNA and the RetroScript Kit (Ambion, Austin, TX) under the conditions recommended by the manufacturer. To clone CYP7A1, the forward primer was 5-AGATCTATGATGACCATCTCTTTGATTTGGGG-3 and reverse primer was 5-AAGCTTCACAGATGTTTCAGTTTATATTTAAACTC-3. These primers were designed based on the available hamster CYP7A1 sequence (the GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”L04690″,”term_id”:”431016″,”term_text”:”L04690″L04690). PCR was performed using 2 l of hamster liver cDNA and the Fail Safe PCR System (Epicentre Technologies, Madison, WI) Buffer E. The target DNA was amplified using 40 cycles of 94C for 30 seconds, 60C for 30 seconds, and 72C for 6 minutes. The 1514 bp product was cloned into a transcription plasmid (T7PA) at the BglII and HindI II sites and confirmed by DNA sequence analysis. To clone CYP46A1, the forward primer was 5-ATGAGCCCCGGGCTGCTGCTG-3 and reverse primer was 5-TCAGCAGGGTGGGGGTGGGG-3. These primers were designed based on the mouse CYP46A1 sequence (the GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_010010″,”term_id”:”1376175715″,”term_text”:”NM_010010″NM_010010). PCR was performed using 2 l of hamster cortex cDNA and Fail Safe Buffer D. The conditions of the amplification were 40 cycles of 94C for 30 seconds, 58C for 30 seconds, and 72C for 6 minutes. The 1503 bp product was cloned using the TA Topo Kit (Invitrogen) and confirmed by sequence analysis. To clone CYP27A1, the forward primer was 5-ATGGCTGCGTGGAGCCGCACG-3 and reverse primer was 5-ATGATCCGGGAGTTTGTGGG-3. These primers were designed based on the mouse CYP27A1 sequence (the GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_024264″,”term_id”:”924181389″,”term_text”:”NM_024264″NM_024264). PCR was performed using 2 l of hamster liver cDNA and Fail Safe Buffer D. The conditions of the amplification were 40 cycles of 94C for 30 seconds, 48C for 30 seconds, and 72C for 5 minutes. The 1230 bp product was cloned using the TA Topo Kit (Invitrogen) and confirmed by sequence analysis. The partial sequences for hamster CYP46A1 and 27A1 have been GDC0853 submitted to the GenBank (accession numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ915127″,”term_id”:”237511754″,”term_text”:”FJ915127″FJ915127 and “type”:”entrez-nucleotide”,”attrs”:”text”:”FJ915128″,”term_id”:”237511756″,”term_text”:”FJ915128″FJ915128, respectively). 2.4. Quantitative Real Time PCRs (RT-QPCR) Synthesis of cDNA was performed in a 20 l reaction using 1 g of total RNA and the reagents in the Taqman Reverse Transcription Reagents GDC0853 Kit (Applied Biosysytems Inc., Foster City, CA). The reaction conditions were as follows: 25C for 10 min, 48C for 30 min, and 95C for 5 min. QPCR amplifications (performed in triplicate) were carried out using 2 l of cDNA, SYBR green primers (see below) and the SYBR Green PCR Grasp Mix (Applied Biosystems Inc.) as specified by the manufacturer. The reaction volume was 25 l and the final concentration of the primers was 900 nM. Relative RT-QPCR assays were performed with 18S RNA as a normalizer, whereas the absolute RT-QPCR was based on the known amounts of a synthetic transcript of the CYP7A1 gene. All PCR assays were run in the ABI Prism 7000 Sequence Detection System. The conditions of the amplification were.