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.
Pagesy
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INSERM U. 223, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
Croissandeau
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Le Dafniet
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Peillon
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Li
J Y
JY
D000236:Adenoma / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D007031:Hypothalamus / Q000502:physiology; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D010903:Pituitary Gland, Anterior / Q000502:physiology* / Q000503:physiopathology; D010911:Pituitary Neoplasms / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D012016:Reference Values; D015183:Restriction Mapping; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D013973:Thyrotropin-Releasing Hormone; D013006:Growth Hormone; D012194:RNA-Directed DNA Polymerase