Measurement of human growth hormone receptor messenger ribonucleic acid by a quantitative polymerase chain reaction-based assay: demonstration of reduced expression after elective surgery.
Studies of GH receptor (GHR) gene expression in human tissues have been hampered by the limited amount of tissue available for analysis and the low sensitivity of conventional methods. We have developed a quantitative reverse transcriptase-PCR assay for measurement of GHR messenger ribonucleic acid levels in small human tissue biopsies. To compensate for sample to sample variation, an internal RNA standard, which differs from the wild-type GHR transcript by only a few nucleotides, was reverse transcribed and amplified together with the GHR transcripts. PCR was carried out using one biotinylated primer to permit the purification of single stranded PCR products on streptavidin-coated microtiter plates. The ratio between the wild-type and mutated transcripts was determined by two separate minisequence reactions in which a primer, annealed immediately 3' of a variable nucleotide, was extended by a single 3H-labeled nucleotide, complementary to either the wild-type or mutated sequence. The assay range was 0.125-8 x 10(5) transcripts/sample, the mean intraassay coefficient of variation was 8.7%, and the lower limit of detection was 0.125 x 10(5) transcripts/sample. GHR messenger ribonucleic acid levels were detectable in small amounts (10-100 ng) of total RNA extracted from adipose tissue, skeletal muscle, and liver. The GHR gene expression in liver was approximately 10-fold higher than that in skeletal muscle, whereas intermediate levels were found in adipose tissue. In nine patients undergoing elective abdominal surgery, GHR gene expression in skeletal muscle was reduced on day 3 after surgery compared to the baseline level. The decrease in GHR gene expression was accompanied by a decrease in skeletal muscle glutamine. This suggests that the postoperative protein catabolism may be caused at least partly by acquired GH insensitivity due to reduced expression of the GHR gene.
Hermansson
M
M
Department of Internal Medicine, Sahlgrenska Hospital, Goteborg, Sweden.
Wickelgren
R B
RB
Hammarqvist
F
F
Bjarnason
R
R
Wennström
I
I
Wernerman
J
J
Carlsson
B
B
Carlsson
L M
LM
D000005:Abdomen / Q000601:surgery*; D000273:Adipose Tissue / Q000378:metabolism; D000368:Aged; D001706:Biopsy; D005260:Female; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000235:genetics*; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D011986:Receptors, Somatotropin