The allele for leucine destroys the BsrI RE site. Similarly, the 331 G A SNP in the promoter product info region was screened by PCR amplification using oligonucleotide primers at an annealing temperature of 55 C and NlaIV RE digestion. The A allele destroys the NlaIV RE site. RE enzymes were Inhibitors,Modulators,Libraries purchased from New England Biolabs. To investi gate the expression status of PR, we conducted real time qRT PCR analysis using PR primers amplifying the tran script portion common to both the PR A and PR B iso forms and specific to the PR B isoform in the P4 exposed and baseline control cultures in samples 2,3 and 6. This analysis showed that both isoforms of PR were expressed in the responder and non responders cell cultures without significant changes upon P4 exposure.
Inhibitors,Modulators,Libraries Statistical analysis The normalized expression data from twelve chips were analyzed jointly to detect genes and or pathways regu lated by P4. The signal intensities of the transcripts in the chips provide a quantitative measure of their relative Inhibitors,Modulators,Libraries abundance. To assess gene expression changes upon P4 exposure, for each transcript, we calculated signal log ratio in each of the six experimental pairs and plotted the average of the six signal log ratios on the y axis against the average signal intensity in twelve chips on the x axis. The signal log ratio is a measure of fold change in expression level for a transcript in the P4 exposed sample relative to its unexposed control. The change is expressed as the log2 ratio. A signal log ratio of 1 is the same as a 2 fold increase and signal log ratio of 1 is the same as a 2 fold decrease in signal Inhibitors,Modulators,Libraries intensity of a tran script.
We assumed that signal intensity of a transcript that is not regulated Inhibitors,Modulators,Libraries by P4 would randomly fluctuate around a mean value on repeated measurements. Therefore the average signal log ratios of the six pairs would randomly distribute on the y axis close to zero. We also assumed that signal intensities of transcripts expressed at similar levels would show similar levels of variations, as strongly suggested in Figure 2, on repeated measurements and therefore their average signal log ratios would also be comparable. However, if a transcript is regulated by P4, its average signal intensity would be displaced towards higher or lower mean values on the x axis and its average signal log ratio would be dispersed further away from zero on the y axis relative to the other transcripts of similar signal intensity, which are not regulated by P4.
We refer to the transcripts with the most extreme average signal log ratios com pared to those transcripts expressed at similar levels as out liers and evaluate them as candidates for regulation by P4. Different approaches to identify the outlier transcripts are conceivable. For example, distribution of signal log ratios for all transcripts can be approximated animal study by continuous curves that define significance boundaries.