Plasma membrane calcium ATPase (PMCA4): a housekeeper for RT-PCR relative quantification of polytopic membrane proteins.
BACKGROUND
Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters.


RESULTS
In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line.


CONCLUSIONS
We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.
Calcagno
Anna Maria
AM
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Bethesda, MD 20892-42546, USA. calcagnoa@mail.nih.gov
Chewning
Katherine J
KJ
Wu
Chung-Pu
CP
Ambudkar
Suresh V
SV
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000941:Antigens / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D053499:Plasma Membrane Calcium-Transporting ATPases; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D000941:Antigens; D000970:Antineoplastic Agents; D000252:Calcium-Transporting ATPases; D027682:Cation Transport Proteins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D007041:Hypoxanthine Phosphoribosyltransferase; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; C026908:ubiquitous tissue antigen; C499076:ATP1A1 protein, human; C421109:multidrug resistance-associated protein 1