GPX5 Myths As Opposed To The Honest Basic Facts
11 Plasma triglycerides were determined by enzymatic colorimetric assays (Genzyme Diagnostics PEI Inc., Charlottetown, PE, Canada). Lyophilized, ground liver samples were SB203580 extracted using an automated solvent extractor (Dionex, Sunnyvale, CA, USA) at 100��C and ?13.8?MPa with 3:1 hexane:2-propanol (method A) or at 125��C and 6.7?MPa with 3:2 hexane:2-propanol (method B). The lipid extracts were analyzed for hepatic triglycerides (Genzyme Diagnostics PEI Inc.), total cholesterol, and free cholesterol by enzymatic methods using kits (Wako Chemicals, Richmond, VA, USA). Feces were collected for 3 consecutive days immediately prior to sacrifice, lyophilized, milled, and stored at??20��C. Fecal lipids were determined by weighing after solvent extraction at 100��C and 13.8?MPa with 3:1 hexane:2-propanol. 2.5. Real-time polymerase chain reaction Total RNA from livers was extracted using a TRIzol Plus RNA purification kit (Invitrogen, Life Technologies, Carlsbad, CA, USA), and cDNA was synthesized using GeneAmp RNA polymerase Vorinostat chain reaction (PCR) kit (Applied Biosystems, Foster City, CA, USA) as per manufacturer��s protocol. Approximately 1?��L of diluted cDNA (1:10) was used in each real-time PCR reaction carried out using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) with an Mx3000P instrument (Stratagene, Cedar Creek, TX, USA). The cycle conditions were as follows: 5 minutes at 95��C, followed by 20�C35 cycles of incubation at 94��C for 15 seconds, and then at 55�C60��C for 1 minute and 72��C for 30 seconds. Sequences of the primers used for this study are shown in Table?2. The primers were validated by assessing the size and sequencing of PCR products. No accumulation of nonspecific products and primer�Cdimers was observed in a gel electrophoresis test of the PCR reaction products. Results were analyzed using the software provided with the Stratagene Mx3000P QPCR system. Differences in mRNA expression were calculated after normalizing to 18S or ��-actin expression. Table?2 Sequences of PCR primers. 2.6. Statistical analysis All data are expressed as means?��?standard error. Differences between control and HPMC groups were determined by two-tailed Student t tests. When variances of each group were unequal, significance of differences was determined using Welch��s test. Pearson��s GPX5 correlation coefficients were calculated for investigating relationships of plasma total cholesterol, plasma adiponectin concentrations, hepatic cholesterol, and triglyceride concentrations with the expression of hepatic genes (JMP 7 statistical program; SAS Institute, Cary, NC, USA). Significance was defined as p?
