Preliminary experiments were performed to determine the appropriate cycle number for amplifying cDNA from each gene product during the exponential phase of PCR (LIF, 34 cycles; cyclophilin, 40 cycles). Serial dilutions of a plasmid standard and uterine cDNA sample were amplified in parallel for LIF and cyclophilin to verify that the plasmid standards and cDNA amplified with similar efficiencies. One uterine sample from each hormone treatment group and five samples from each stage of pregnancy were amplified for LIF, and the identity of the PCR products was verified by Southern blot analysis. Samples from the pregnancy series were also subjected to PCR for cyclophilin, and the product was verified by blot. Additionally, 5 aliquots of one uterine sample from each stage of pregnancy and treatment group were reverse transcribed and amplified to assess intraassay variability of the entire RT-PCR procedure. proventil inhaler
The quantitative PCR assay was based upon a procedure developed to quantify growth factors in ovarian tissue. After reverse transcription, all samples were diluted to 3.3 volumes with sterile water containing yeast RNA at a final concentration of 10 ng/^l. All subsequent dilutions of cDNA and standards included this concentration of carrier RNA. Plasmid DNA containing LIF or cyclophilin subclones were used to generate standard curves for both compounds.