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. 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.
Roberts-Thomson
S J
SJ
School of Pharmacy, The University of Queensland, QLD 4072, St. Lucia, Australia.
Holman
N A
NA
May
F J
FJ
Lee
W J
WJ
Monteith
G R
GR
D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D027682:Cation Transport Proteins; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D006801:Humans; D053499:Plasma Membrane Calcium-Transporting ATPases; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C506952:ATP2B1 protein, human; D027682:Cation Transport Proteins; D017931:DNA Primers; D015342:DNA Probes; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases