Quantitative analysis of growth hormone (GH) pre-mRNA expression in cultured rat anterior pituitary cells by an intron-specific and competitive PCR method.
We have developed a novel method of quantifying growth hormone(GH) pre-mRNA expression in anterior pituitary cells. DNA-free total RNA extracted from cultured rat anterior pituitary cells was reverse transcribed(RT) to cDNA, and RT products were subsequently quantitated by competitive PCR using intron-specific primers of rat GH gene. After 6-h of incubation in treated cells, dexamethasone(Dex) and triiodo-L-thyronine(T3) significantly increased GH pre-mRNA levels(3.2- and 2.2-fold compared to non-treated cells, respectively). However, Northern blot analysis did not detect significant changes in GH mRNA levels. After 24-h incubation with Dex and T3, significant increases in GH mRNA levels were detected on Northern blots, but GH pre-mRNA levels did not differ between treated and non-treated cells. These findings suggest that both Dex and T3 treatments rapidly increase GH pre-mRNA levels in normal somatotropes. This method has high sensitivity and widespread application to the analysis of pre-mRNAs of target genes.
Matsubara
S
S
First Department of Internal Medicine, Kagawa Medical University, Japan.
Sato
M
M
Ohye
H
H
Murao
K
K
Takahara
J
J
D000818:Animals; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D013006:Growth Hormone / Q000235:genetics*; D007438:Introns / Q000235:genetics; D008297:Male; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014158:Transcription, Genetic; D014284:Triiodothyronine / Q000494:pharmacology
D005938:Glucocorticoids; D012322:RNA Precursors; D012333:RNA, Messenger; D014284:Triiodothyronine; D003907:Dexamethasone; D013006:Growth Hormone