Plasma membrane calcium ATPase (PMCA4): a housekeeper for RT-PCR relative quantification of polytopic membrane proteins.
Plasma membrane calcium ATPase (PMCA4): a housekeeper for RT-PCR relative quantification of polytopic membrane proteins.
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DOI：
10.1186/1471-2199-7-29
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发表时间：
2006-09-17
影响因子：
--
通讯作者：
Ambudkar SV
中科院分区：
文献类型：
--
作者：
Calcagno AM;Chewning KJ;Wu CP;Ambudkar SV
虽然实时RT-PCR数据的相对量化可以提供有价值的信息，但一个限制仍然是选择适当的参考基因。目前还没有一个基因成为通用的参考基因，而且围绕一些更常用的参考基因也存在很大争议，例如甘油醛-3-磷酸脱氢酶(GAPDH)。此时，没有编码质膜蛋白的基因作为参考基因，质膜蛋白的相对定量是用编码可溶性蛋白的基因进行的，这些基因在数量和靶向性和转运方面与质膜蛋白有很大的不同。在这项工作中，我们的目标是识别一个看家基因，理想情况下是一个编码质膜蛋白的基因，无论药物治疗如何，它的表达都保持不变，并在广泛的组织中用于相对定量ATP结合盒(ABC)质膜转运体的实时RT-PCR数据。在评估两个编码可溶性蛋白的常用参考基因和两个编码膜蛋白的基因的表达水平的研究中，一个质膜蛋白，质膜钙-ATPase 4(PMCA4)，与两个已在使用的参考基因的表达水平相当。此外，PMCA4在八种药物处理的细胞系中的表达变化很小，并被发现优于常用的参考基因GAPDH和HPRT1。最后，我们展示了PMCA4作为一个参考基因用于在耐药的肺癌细胞系中正常化ABC转运蛋白的表达。我们发现PMCA4是一个很好的管家基因，可以使包括转运蛋白和受体在内的多角体膜蛋白的基因表达正常化。
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. 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. 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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影响因子：
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