Semiquantitative RT-PCR with 25 amplification cycles for the FSHp, I/A pB subunit, and p-actin and with 30 cycles for FS cDNAs were conducted as described previously. The strategies used for determination of amplification cycles for the GnRH-R and calmodulin cDNAs were essentially the same. Briefly, 4 ^l of cDNA mixture was amplified for 15 to 35 cycles under the identical conditions given in the previous papers. Based upon the exponential accumulation of radioactive PCR products, 30 amplification cycles were optimized with 0.2 ^g total RNA for these two sets of primers. For each gene studied, the mRNA level of each sample was normalized to that of p-actin mRNA, which was determined simultaneously under identical conditions except that different primers were used.
RNase Protection Assay (RPA)
Hybridizations and RNase digestions were performed with an RPA-II kit from Ambion Inc. (Austin, TX) under conditions described previously. Relative intensities of protected fragments were determined by transmittance scanning densitometry and were normalized with the protected fragment for an antisense, porcine p-actin ribo-probe. antibiotics levaquin
The mRNA level of each gene of interest was adjusted first to a constant amount of p-actin mRNA. Student’s t-test was used to determine differences between the high and low FSH groups. The correlative relationship between any two variables was determined by correlation analysis. The data are presented as the mean ± SEM.