Measurement of PTH/PTHrP Receptor mRNA Using a Competitive Internal Standard.

Abstract

Parathyroid hormone (PTH) /PTH-related peptide receptor (PTH/PTHrPR) mRNA is widely distributed in rat tissues. In order to quantify PTH/PTHrPR in various tissues and to elucidate the effect of estrogen on PTH/PTHrPR mRNA transcription, a simple and reliable polymerase chain reaction (PCR) -based method for quantifying rat PTH/PTHrPR mRNA was developed. This method involves co-amplification of cDNA transcribed from sample RNA with the internal standard synthesized by insertion of a DNA fragment near the midportion of the rat target PTH/PTHrPR cDNA to reduce tube-to-tube amplification variations. The reverse transcription rate and the coefficient of variation of this assay were 22% and 12.0%, respectively. PTH/PTHrPR mRNA levels in the kidney removed from 12 week old rats were 165.8± 20.7 × 10-19mol/g total RNA (n=4) and was higher than those in any other tissues. The chronic administration of estrogen significantly increased PTH/PTHrPR mRNA levels in the uterus, but had no significant effects on those in the kidney, heart, lung or brain. However, in the in vitro experiment using rat osteosarcoma ROS 17/2.8 cells, the administration of estrogen at a dose of 10-8M significantly decreased PTH/PTHrPR mRNA levels. These findings suggest that the expression of PTH/PTHrPR is under the control of estrogen in certain tissues.