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Type: Journal article
Title: Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase-polymerase chain reaction
Author: Anderson, P.
O'Loughlin, P.
May, B.
Morris, H.
Citation: Journal of Molecular Endocrinology, 2003; 31(1):123-132
Publisher: Soc Endocrinology
Issue Date: 2003
ISSN: 0952-5041
Statement of
PH Anderson, PD O'Loughlin, BK May and HA Morris
Abstract: Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.
Keywords: Kidney; Animals; Rats; Rats, Sprague-Dawley; Calcium, Dietary; Calcitriol; Parathyroid Hormone; Cytochrome P-450 Enzyme System; Steroid Hydroxylases; 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; DNA, Complementary; RNA, Messenger; DNA Primers; Sensitivity and Specificity; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Base Sequence; Vitamin D3 24-Hydroxylase
Rights: © 2003 by Society for Endocrinology
RMID: 0020030038
DOI: 10.1677/jme.0.0310123
Appears in Collections:Molecular and Biomedical Science publications

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