Quantitative Gonadotropin-Releasing Hormone: RESULTS(1)
RT-PCR was employed to increase the sensitivity of detection, and a sequence of 399 bp of GnRH mRNA was amplified in all the endometrial samples analyzed from fertile women in both the follicular and luteal phases of the menstrual cycle. buy ortho tri-cyclen online
The localization of the expected fragment (524 bp) for the GnRH receptor mRNA was also examined in the endometrial samples from the same patients, and amplified signal was detected using one round of PCR (40 cycles) in 30% of the samples studied. For this reason, the sensitivity was further increased by performing two rounds of nested PCR (30 cycles in the first round and 35 in the second) in order to amplify a signal. Electrophoresis of RT-PCR fragments of each sample produced a 231-bp size band corresponding to the fragment produced by the specific primer used in the second round PCR. By this method we were able to detect GnRH receptor in all the samples studied throughout the different phases of the menstrual cycle (Fig. 3).
p-Actin mRNA expression was ascertained in all the samples studied, thus confirming the integrity of the RNA and the RT-PCR process.
FIG. 1. a) Construction of the target cDNA by amplification of a 399-bp fragment from cDNA obtained after RT of 1 ^g total RNA extracted from human luteal endometrium. Primers used for this step were 3′ and 5′ GnRH (Table 1). b) Creation of an artificial deletion in the target cDNA from a. By amplification of the target cDNA with the 5′ GnRH primer and the 3’C GnRH floating primer (Table 1), a 195-bp deletion was obtained. c) Sequence of the competitive cDNA for GnRH. The cDNA sequence is identical with the target cDNA except for the 195-bp deletion created in b. The sequences of the primer binding sites for GnRH 3′ and GnRH 5′ are identical for the target cDNA and native cDNA, suggesting that in a coamplification of either of these cDNAs with the competitive cDNA, the cDNAs will truly compete for the primers and for the polymerase and will be amplified with the same affinity.
FIG. 2. The upper panel shows a 2% agarose gel stained with ETB. Declining amounts of target cDNA were coamplified with 30 amol competitive cDNA. The lower panel shows the standard curve obtained from this gel. The log ratio of target to competitive product density was plotted against the log amount of target initially added to the PCR.
FIG. 3. GnRH receptor mRNA expression in the different phases of the menstrual cycle: 1) Positive control (2nd trimester human placenta); 2) Negative control; 3) midproliferative endometrium; 4) late proliferative endometrium; 5) early luteal endometrium; 6) midluteal endometrium; 7) late luteal endometrium.