RNA Isolation and Preparation
Total RNA was isolated from the remaining half of each pituitary gland by guanidine isothiocyanate extraction and CsCl centrifugation as described by Chirgwin et al.. Prior to use, RNA was treated with RNase-free DNase I at 37°C for 30 min. After extraction with phenol-chloroform and ethanol precipitation, the RNA was redissolved into diethyl pyrocarbonate-treated water. buy cheap antibiotics
Primers Used for Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Primers used for porcine FSHp, I/A pB subunit, FS, and p-actin cDNA amplifications were the same as described previously. Based on the partial calmodulin cDNA sequence (290 base pairs [bp]) cloned from WC pituitary RNA by differential-display PCR in our laboratory, two primers with sequences of 5′-TGACAAGGA-TGGCAATGG-3′ for the sense and 5′ -CGGGAAAAAG-GAGTTGAAAG-3′ for the antisense were designed. The expected PCR product size amplified by this set of primers is 199 bp. Sense and antisense primers for the GnRH-R were 5′-GCTGCCTCTTCATTATCC-3′ and 5′-AGA-CTGTGGGACAAATGG-3′, which correspond to regions 668-675 and 906-923 nucleotides (nt) of the pig GnRH-R sequence, respectively. The amplified PCR product size was expected to be 255 bp. All oligonucleotides used in this study were synthesized with an Oligo 1000 DNA Synthesizer (Beckman Instruments, Palo Alto, CA).