Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line

The plasma membrane Ca 2+ pump is a key regulator of cytosolic free Ca 2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca 2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease...

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Published in:	Journal of pharmacological and toxicological methods Vol. 44; no. 3; pp. 513 - 517
Main Authors:	Roberts-Thomson, S.J, Holman, N.A, May, F.J, Lee, W.-J, Monteith, G.R
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-11-2000
Subjects:	Breast Neoplasms - enzymology Breast Neoplasms - genetics Calcium-Transporting ATPases - genetics Calcium-Transporting ATPases - metabolism Cation Transport Proteins DNA Primers - chemistry DNA Probes - chemistry DNA, Neoplasm - analysis Expression Female Humans MCF-7 Plasma Membrane Calcium-Transporting ATPases Plasmalemmal Ca 2+ pump PMCA Real-time RT-PCR Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger - metabolism Tumor Cells, Cultured Real-time RT-PCR Plasmalemmal Ca 2+ pump Expression MCF-7 PMCA
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Summary:	The plasma membrane Ca 2+ pump is a key regulator of cytosolic free Ca 2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca 2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca 2+ ATPase (PMCA1) isoform of the plasma membrane Ca 2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1056-8719 1873-488X
DOI:	10.1016/S1056-8719(01)00112-5