β-2 microglobulin is unsuitable as an internal reference gene for the analysis of gene expression in human colorectal cancer
β-2 microglobulin is unsuitable as an internal reference gene for the analysis of gene expression in human colorectal cancer
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DOI：
10.3892/br.2013.53
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发表时间：
2013-03-01
影响因子：
2.3
通讯作者：
Okazumi, Shinichi
中科院分区：
文献类型：
--
作者：
Nihon-Yanagi, Yasuhiro;Terai, Kensuke;Okazumi, Shinichi
众所周知，基因表达水平应标准化为仔细选择的和适当稳定的内部对照基因。然而，许多研究表明，管家（HK）基因，通常用作内部控制基因的表达变化很大。许多研究表明，β-2微球蛋白（B2 M），HK基因，经常被用作内部参考基因，在结肠直肠癌组织中表达水平低，当使用实时逆转录酶-聚合酶链反应（RT-PCR）进行评估。由于各种HK基因的表达水平根据组织类型或实验条件而变化的事实，已经建议对于某些组织应平行分析几种对照基因。在本研究中，使用市售工具，相对于三个HK基因，β-葡萄糖醛酸酶（GUS），β-肌动蛋白（BA）和B2 M，分析了散发性人结直肠癌和非癌组织中Toll样受体2（TLR 2）和4（TLR 4）的mRNA表达水平。使用三种基因单独和一起定量相对表达水平，并比较癌性和非癌性结直肠组织标本中TLR 2和TLR 4的表达。以GUS和BA为内对照基因，在大多数情况下获得了一致的数据。当B2 M被用作内部对照基因时，TLR 2和TLR 4的表达被证明在癌性结直肠组织中高于非癌性结直肠组织。这些结果与先前观察到的结直肠癌组织中B2 M低水平表达一致，并表明B2 M可能不适合作为结直肠癌基因表达研究的内部对照基因。
It is well-known that gene expression levels should be normalized to a carefully selected and appropriately stable internal control gene. However, numerous studies have demonstrated that the expression of housekeeping (HK) genes, typically used as internal control genes varies considerably. A number of studies have shown that beta-2 microglobulin (B2M), an HK gene, frequently used as an internal reference gene, is expressed at low levels in colorectal cancer tissue, when assessed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Due to the fact that the expression levels of various HK genes vary depending on the tissue type or experimental conditions, it has been suggested that several control genes should be analyzed in parallel for certain tissues. In the present study, mRNA expression levels of toll-like receptors 2 (TLR2) and 4 (TLR4) in sporadic human colorectal cancerous and non-cancerous tissues were analyzed relative to three HK genes, beta-glucuronidase (GUS), beta-actin (BA) and B2M, using a commercially available tool. Relative expression levels were quantified using the three genes individually and together, and TLR2 as well as TLR4 expression was compared in cancerous and non-cancerous colorectal tissue specimens. Consistent data were obtained in most cases when GUS and BA were used as internal control genes. When B2M was used as the internal control gene, TLR2 and TLR4 expression was demonstrated to be higher in cancerous compared to non-cancerous colorectal tissues. These results were consistent with previous observations of low-level B2M expression in cancerous colorectal tissue and suggest that B2M may be inappropriate as an internal control gene for gene expression studies of colorectal cancer.