MgCl2 concentrations were reoptimized using the LightCycler protocol (Roche Molecular Biochemicals, Mannheim, Germany). Preliminary real-time RT-PCR experiments were performed with each primer pair to determine the annealing temperature that yielded www.selleckchem.com/products/mek162.html the greatest amount of specific product with melting temperature separable from primer-dimer temperature. Relative standard curves and PCR assay conditions Standard curves were prepared for each run using known quantities of cDNA (10-fold dilutions beginning at 15 ng/��l) and primers for the gene of interest or ��-actin. Light Cycler-Fast Start DNA Master SYBR Green I reaction mix containing 0.4 ��M forward and reverse primers, 4 M MgCl2, and 1 ��l LightCycler-Fast start DNA master SYBR Green 1 dye was used for quantitative real-time PCR analysis according to the manufacturer’s protocols (Roche Molecular Biochemicals, Mannheim, Germany).
A volume of 1 ��l from a 10-fold dilution of cDNA was added as the PCR template. A no-target control received 9 ��l reaction mix and 1 ��l water. PCR amplification was performed in triplicate wells. A four-step experimental run protocol was performed: i) denaturation program (10 min at 95��C); ii) 45 cycles of four-segment amplifications consisting of denaturation at 95��C for 10 s, annealing (60�C56��C) for 5 s, extension at 72��C for 10 s, and data acquisition at 83��C for 1 s. A temperature transition rate of 2��C/s with a single fluorescence measurement was used. iii) Melting curve program (50�C95��C, with heating rate of 0.1��C/s up to 98��C with continuous fluorescence measurement); and vi) a cooling program down to 40��C.
Relative quantitation measurements were performed using external standard curves for both target and ��-actin housekeeping gene. The second derivative maximum method was used for cycle threshold (ct) calculation from amplification curves. The respective concentration for any given sample was calculated using crossing-cycle analysis provided by the LightCycler software. Statistics Microarray data analysis was performed with the Partek genomic suite software suite 6.3 (Partek, MO). Probe set data were summarized, background adjusted, and quantile normalized using robust multichip average methodology described in Irizarry et al. (20). A false discovery rate correction was applied to post hoc pairwise group comparisons of Pearson’s correlation coefficients obtained by ANOVA.
Heat map and clustering of the gene expression data were also generated using Partek. P �� Brefeldin_A 0.05 was considered significant, with an arbitrary threshold of 1.5-fold difference between the four groups analyzed simultaneously. As the mRNA expression in four animals from each of the four diet groups was separately analyzed, four comparisons were possible when comparing the effects of TFA diet animals with respect to controls receiving standard chow and the effect of the addition of MSG to both diets.