Rapid Simultaneous Measurement of Ratα- and Thyrotropin (TSH)β-Subunit Messenger Ribonucleic Acids (mRNAs) by Solution Hybridization: Regulation of TSH Subunit mRNAs by Thyroid Hormones*

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Abstract
Using a sensitive method for the simultaneous measurement of TSH subunit mRNA, their hormonal regulation has been investigated in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat .alpha.- and TSH .beta.-subunit mRNA were synthesized. These probes were 5''-end labeled with .gamma.-[32P] ATP and hybridized with total pituitary RNA obtained from T3 treated [triiodothyronine]-and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNA would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography and densitometry permits quantification of the mRNA. Both rat .alpha. and TSH.beta. mRNA were detected with as little as 0.1 .mu.g total pituitary RNA, representing a > 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNA in normal and hypothyroid animals. In all treatment groups, the TSH.beta. mRNA was affected to a greater extent than the .alpha. mRNA by the changes in thyroid status. The ratio of .alpha.- to .beta.-subunit mRNA was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNA from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNA encoding TSH subunits specifically and other pituitary proteins in general.