Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated by development and inflammation, but not by iron status or erythropoietin

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4308-4310 ◽  
Author(s):  
Jan Krijt ◽  
Martin Vokurka ◽  
Ko-Tung Chang ◽  
Emanuel Nečas
Keyword(s):  
Polymerase Chain Reaction ◽  
Iron Overload ◽  
Real Time ◽  
Postnatal Development ◽  
Iron Status ◽  
Mrna Level ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Juvenile Hemochromatosis ◽  
Polymerase Chain

Abstract Mutations of hepcidin (HAMP) and hemo-juvelin (HJV) genes have been recently demonstrated to result in juvenile hemochromatosis. Expression of HAMP is regulated by iron status or infection, whereas regulation of HJV is yet unknown. Using quantitative real-time polymerase chain reaction, we compared expression of Hamp and Rgmc (the murine ortholog of HJV) in livers of mice treated with iron, erythropoietin, or lipopolysaccharide (LPS), as well as during fetal and postnatal development. Iron overload increased Hamp expression without effect on Rgmc mRNA. Erythropoietin decreased Hamp mRNA, but Rgmc expression was unchanged. Hamp mRNA level decreased after birth by 4 orders of magnitude, without significant changes in Rgmc expression. Administration of LPS elevated Hamp mRNA levels, while markedly decreasing hepatic Rgmc mRNA levels (to ∼5% after 6 hours). The responses of Hamp and Rgmc were quite different and suggested that human HJV expression could be modulated by inflammation.

scholarly journals An Inexpensive Gel Electrophoresis-Based Polymerase Chain Reaction Method for Quantifying mRNA Levels

2005 ◽  
Vol 4 (2) ◽  
pp. 157-168 ◽  
Author(s):  
William D. Bradford ◽  
Laty Cahoon ◽  
Sara R. Freel ◽  
Laura L. Mays Hoopes ◽  
Todd T. Eckdahl
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Polymerase Chain Reaction Method ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Teaching And Research ◽  
Undergraduate Institutions ◽  
Polymerase Chain

In order to engage their students in a core methodology of the new genomics era, an everincreasing number of faculty at primarily undergraduate institutions are gaining access to microarray technology. Their students are conducting successful microarray experiments designed to address a variety of interesting questions. A next step in these teaching and research laboratory projects is often validation of the microarray data for individual selected genes. In the research community, this usually involves the use of real-time polymerase chain reaction (PCR), a technology that requires instrumentation and reagents that are prohibitively expensive for most undergraduate institutions. The results of a survey of faculty teaching undergraduates in classroom and research settings indicate a clear need for an alternative approach. We sought to develop an inexpensive and student-friendly gel electrophoresis-based PCR method for quantifying messenger RNA (mRNA) levels using undergraduate researchers as models for students in teaching and research laboratories. We compared the results for three selected genes measured by microarray analysis, real-time PCR, and the gel electrophoresis-based method. The data support the use of the gel electrophoresis-based method as an inexpensive, convenient, yet reliable alternative for quantifying mRNA levels in undergraduate laboratories.

Real-Time Reverse Transcriptase Polymerase Chain Reaction: An Improvement in Detecting mRNA Levels in Mouse Cranial Tissue

2006 ◽  
Vol 117 (7) ◽  
pp. 2227-2234
Author(s):  
Rashmi Singh ◽  
Ren?? F. Recinos ◽  
Michael Agresti ◽  
Richard B. Schaefer ◽  
Mark Bosbous ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals 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

2003 ◽  
Vol 31 (1) ◽  
pp. 123-132 ◽  
Author(s):  
PH Anderson ◽  
PD O'Loughlin ◽  
BK May ◽  
HA Morris
Keyword(s):  
Vitamin D ◽  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Vitamin D Receptor ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Polymerase Chain

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.

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