Each reaction was repeated five times. The PCR products were separated on 2.5% agarose-TAE (40 mM Tris-acetate, 1 mM EDTA) gels containing ethidium bromide (10 mg/ml) and visualized by UV light. Representative RT-PCR products were excised from agarose gels and confirmed by DNA sequencing. Expression of HOXC10, HOXC11, HOXD10, HOXD11, and G3PDH were assessed on unsaturated gels by densitometric quantification by using laser densitometry, and HOX values were normalized to G3PDH. RT-PCR values are presented as a ratio of the specified gene’s signal in the selected linear amplification cycle divided by the G3PDH signal.
The gels presented in the figures are loaded for illustrative purposes and contain more PCR product than used for quantification.
The intensity of the bands was quantified by densitometry, and results were expressed as the ratio of intensity of HOXC10, HOXC11, HOXD10, and HOXD11 relative to G3PDH in all samples. Data were analyzed with the SigmaStat (Jandel Scientific, San Rafael, CA) software package. The values were expressed as mean ± SEM. Student /-test was used to compare results of HOX gene expression from endometrial biopsy samples representing proliferative and secretory phases. The differences between treatment groups in experiments with either stromal or Ishikawa cells were analyzed by one-way ANOVA. A statistically significant difference was defined as P < 0.05.