Data generated by real-time PCR were compiled and collected using SDS 2.2 software (Applied Biosystems). Data were exported to QGene to determine the PCR amplification efficiency (E) for each primer pair where E = 10(-1/slope) as determined by linear regression analysis of a dilution series of reactions [[38 (link)]; see Results]. All amplifications had a PCR efficiency value between 1.9 and 2.2. To normalize data for geNorm analysis the efficiency of each primer pair (E), together with the Ct values, was used to calculate a relative gene expression value for each transcript using the equation E ΔCt(Min Ct-Ct sample) where Min Ct is the lowest Ct value for the primer pair and Ct sample is the Ct value for each amplification [10 (link),34 (link)]. The Ct is defined as the number of cycles needed for the fluorescence to reach a specific threshold level of detection and is inversely correlated with the amount of template present in the reaction [39 (link)]. The relative stability of the eight reference genes was then calculated using geNorm [34 (link)]. This program evaluates a gene expression stability measure (M) for each reference gene by calculating pair-wise variations with all other control genes and ranks them in order of increasing expression stability. Statistical analysis of Ct value differences was performed using the Sigma-Stat 3.5 package (Aspire Software, Leesburg, VA). Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey method for pair-wise multiple comparisons. Student's t-test was used to compare differences in mean Ct values between male and female tissues. Student's t-test was also used to determine significant differences in expression following chemical treatment. Vehicle (DMSO, EtOH) was compared to untreated and all other chemicals (E2, T, ICI, BNF, TCDD) were compared to the vehicle of preparation (DMSO). Significance was set at P < 0.05.
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