Calcagno Anna Maria, Chewning Katherine J, Wu Chung-Pu, Ambudkar Suresh V
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Bethesda, MD 20892-42546, USA.
BMC Mol Biol. 2006 Sep 17;7:29. doi: 10.1186/1471-2199-7-29.
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.
尽管实时逆转录聚合酶链反应（RT-PCR）数据的相对定量可提供有价值的信息，但一个限制仍然是选择合适的内参基因。没有一个基因已成为通用的内参基因，围绕一些更常用的内参基因，如甘油醛-3-磷酸脱氢酶（GAPDH），存在很多争议。目前，没有编码质膜蛋白的基因用作内参基因，质膜蛋白的相对定量是用编码可溶性蛋白的基因进行的，这些可溶性蛋白在数量、靶向和运输方面与质膜蛋白有很大差异。在本研究中，我们的目的是鉴定一个管家基因，理想情况下是一个编码质膜蛋白的基因，其表达在药物处理和广泛的组织中保持不变，用于ATP结合盒（ABC）质膜转运蛋白实时RT-PCR数据的相对定量。
在评估两个编码可溶性蛋白的常用内参基因和两个编码膜蛋白的基因的表达水平的研究中，一种质膜蛋白，即质膜钙ATP酶4（PMCA4），与已使用的两个内参基因相当。此外，PMCA4的表达在八种药物处理的细胞系中变化很小，并且发现优于常用的内参基因GAPDH和HPRT1。最后，我们展示了PMCA4用作内参基因来标准化耐药肺癌细胞系中ABC转运蛋白的表达。
我们发现PMCA4是用于多聚体膜蛋白（包括转运蛋白和受体）基因表达标准化的良好管家基因。
