Quantitative analysis by polymerase chain reaction of growth hormone receptor gene expression in human liver and muscle.
A single form of GH receptor (GHR) messenger RNA (mRNA) of 4.5 kilobases, encoding the full-length GHR, has been found in man. To measure the absolute number of mRNA molecules encoding the GHR in human tissues, we developed a quantitative polymerase chain reaction assay. An internal control RNA was constructed by inserting a 50-basepair fragment of the rat PRL receptor complementary DNA into a portion of the human GHR complementary DNA. The internal control RNA and the target mRNA were amplified together with the same set of primers. Twenty-four cycles of amplification were used to satisfy an exponential phase of amplification. It was possible to detect as few as 500 molecules of target mRNA/micrograms total RNA. In 3 liver samples obtained from normal donors at the time of transplant, the amount of GHR mRNA ranged from 0.5 +/- 0.1 to 1.4 +/- 0.4 x 10(6) molecules/micrograms total RNA. These results were confirmed by slot blot analysis of the same samples. The number of receptor transcripts did not appear to be correlated with the receptor-binding capacity found in the 3 liver samples. In 7 muscle biopsies, GH receptor mRNA varied between 4.0 +/- 0.4 and 34.6 +/- 1.4 x 10(4) molecules/micrograms total RNA. This technique allows direct measurement of GHR gene expression in human tissues and represents a valuable tool, particularly for tissues such as muscle, in which the receptor protein cannot be measured using conventional binding assays.
Martini
J F
JF
INSERM U-344, Endocrinologie Moléculaire, Necker-Enfants Malades, Paris, France.
Villares
S M
SM
Nagano
M
M
Delehaye-Zervas
M C
MC
Eymard
B
B
Kelly
P A
PA
Postel-Vinay
M C
MC
D001483:Base Sequence; D015139:Blotting, Southern; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011986:Receptors, Somatotropin / Q000235:genetics*; D012016:Reference Values
D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D012313:RNA