Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.
Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR.
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DOI：
10.1186/1471-2199-7-33
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发表时间：
2006-10-06
影响因子：
--
通讯作者：
Thein SL
中科院分区：
文献类型：
--
作者：
Silver N;Best S;Jiang J;Thein SL
通常被称为管家基因的控制基因经常用于使不同样品之间的mRNA水平正常化。然而，这些基因的表达水平可能在组织或细胞之间变化，并且可能在某些情况下发生变化。因此，管家基因的选择是基因表达研究的关键。为了解决这一问题，在分离的人网织红细胞中研究了7个候选管家基因，包括几个常用的管家基因。为此，通过比较每个样品内“基因对”的相对表达，采用简单的ΔCt方法。在此基础上，根据31个样本中基因表达差异的重复性对候选管家基因的稳定性进行排序。表达模式的初步筛选表明，7个基因中的1个在网织红细胞中以非常低的水平表达，并从进一步分析中排除。其他6个基因的表达稳定性范围（从最稳定到最不稳定）为：GAPDH（甘油醛3-磷酸脱氢酶）、SDHA（琥珀酸脱氢酶）、HPRT 1（次黄嘌呤磷酸核糖转移酶1）、HBS 1 L（HBS 1样蛋白）和AHSP（α血红蛋白稳定蛋白），其次是B2 M（β 2-微球蛋白）。使用这种简单的方法，GAPDH被发现是最合适的管家基因在网织红细胞中的表达研究，而常用的B2 M应避免。
Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.
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