Quantitative analysis of estrogen receptor mRNA in human endometrium throughout the menstrual cycle using a real-time reverse transcription-polymerase chain reaction assay

We conducted a quantitative analysis of ERalpha and ERbeta mRNA expression in normal human endometrium throughout the menstrual cycle in regular menstruating premenopausal women, taking advantage of this real-time PCR assay. Endometrial dating was determined from the histology of the endometrium and classified into: proliferative endometrium and secretory endometrium. Both ERalpha and ERbeta mRNA expression were detected in all endometrial samples at both proliferative and secretion phase. However ERalpha mRNA expression level was higher than that of ERbeta specially during proliferative phase. These results suggest that estrogenic effects occur predominantly through ERalpha than ERbeta.     

Detection of thyrotropin-releasing hormone (TRH) mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary.

To investigate the presence of TRH mRNA in the human anterior pituitary tissue, total RNA from human normal and tumoral anterior pituitary, hypothalamus (positive control) and muscle tissues (negative control) was reverse transcribed (RT) to the first strand of cDNA. RT products were then amplified by polymerase chain reaction (PCR) using a set of three exon-specific primers (two external 5' and 3' primers and one internal 3' primer) for a target sequence of the TRH gene including an intronic sequence of about 650 base pairs (bp). Southern analysis of the RT-PCR products specifically hybridizing with a 45-mer TRH probe showed two bands of the predicted sizes (399 and 351 bp) far more intense in hypothalamus than in normal and tumoral anterior pituitary tissue. The 399 and 351 bp RT-PCR products contained the BglII enzyme restriction site included in the TRH cDNA sequences spanned by the primers and the two respective digested fragments which were, as predicted, 337 and 289 bp long, hybridized with the TRH probe. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target TRH sequences in the human normal and tumoral anterior pituitary tissue as well as in the hypothalamus. Our data imply TRH gene expression in the human anterior pituitary.