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Plasma membrane calcium ATPase (PMCA4): a housekeeper for RT-PCR relative quantification of polytopic membrane proteins.

Calcagno AM, Chewning KJ, Wu CP, Ambudkar SV - BMC Mol. Biol. (2006)

Bottom Line: 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.We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Bethesda, MD 20892-42546, USA. calcagnoa@mail.nih.gov

ABSTRACT

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.

Conclusion: We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.

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Related in: MedlinePlus

Standard curve to evaluate efficiency of RT-PCR reaction. The HPRT1 (blue circle) and PMCA4 (red diamond) primers were evaluated over a range of total RNA concentrations (30 ng-300 ng total RNA). The average crossing point values are given for the n = 4, and the error bars represent ± SD.
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Figure 1: Standard curve to evaluate efficiency of RT-PCR reaction. The HPRT1 (blue circle) and PMCA4 (red diamond) primers were evaluated over a range of total RNA concentrations (30 ng-300 ng total RNA). The average crossing point values are given for the n = 4, and the error bars represent ± SD.

Mentions: To determine the optimal concentration of total RNA to use within our studies, the efficiency of the RT-PCR reaction was evaluated over a concentration range. The standard curve for two primers for the detection of HPRT1 and PMCA4 indicates that the efficiency is nearly 2 (Fig. 1). Two hundred nanograms of total RNA was found to be within the linear range of the reaction, and this quantity was used to evaluate the primers for this and all subsequent studies comparing the four genes.


Plasma membrane calcium ATPase (PMCA4): a housekeeper for RT-PCR relative quantification of polytopic membrane proteins.

Calcagno AM, Chewning KJ, Wu CP, Ambudkar SV - BMC Mol. Biol. (2006)

Standard curve to evaluate efficiency of RT-PCR reaction. The HPRT1 (blue circle) and PMCA4 (red diamond) primers were evaluated over a range of total RNA concentrations (30 ng-300 ng total RNA). The average crossing point values are given for the n = 4, and the error bars represent ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1586022&req=5

Figure 1: Standard curve to evaluate efficiency of RT-PCR reaction. The HPRT1 (blue circle) and PMCA4 (red diamond) primers were evaluated over a range of total RNA concentrations (30 ng-300 ng total RNA). The average crossing point values are given for the n = 4, and the error bars represent ± SD.
Mentions: To determine the optimal concentration of total RNA to use within our studies, the efficiency of the RT-PCR reaction was evaluated over a concentration range. The standard curve for two primers for the detection of HPRT1 and PMCA4 indicates that the efficiency is nearly 2 (Fig. 1). Two hundred nanograms of total RNA was found to be within the linear range of the reaction, and this quantity was used to evaluate the primers for this and all subsequent studies comparing the four genes.

Bottom Line: 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.We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Bethesda, MD 20892-42546, USA. calcagnoa@mail.nih.gov

ABSTRACT

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.

Conclusion: We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.

Show MeSH
Related in: MedlinePlus