Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression
Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression
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DOI：
10.1007/s00427-012-0420-x
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发表时间：
2012-11-01
影响因子：
2.4
通讯作者：
Redies, Christoph
中科院分区：
文献类型：
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作者：
Lin, Juntang;Redies, Christoph
内务基因被广泛用作基因表达归一化的内部对照，用于western blotting、northern blotting、RT-PCR等。它们通常被认为在生物体的所有细胞中以相似的水平表达，因为假设这些基因作为构成基因是维持基本细胞功能所必需的。然而，实时RT-PCR实验显示，它们的表达可能因发育阶段、检查组织类型、实验条件等而异。到目前为止，还没有关于它们在胚胎发育中的表达的组织学数据。本研究采用切片和全载原位杂交结合RT-PCR的方法，比较了两种常用管家基因GAPDH和β -actin在发育中的鸡胚的组织学表达谱。我们的研究结果表明，GAPDH mRNA和β -actin mRNA在所有细胞类型或组织中都没有高水平表达。引人注目的是，在一些器官中表达水平非常低。此外，这两个基因在肝脏、血管系统和消化道中表现出部分互补的表达模式。例如，GAPDH在肝脏中的表达比β -肌动蛋白更强烈，但在动脉中的表达水平较低。反之亦然，β -肌动蛋白在砂囊中的表达比GAPDH更强烈，但在心肌细胞中几乎不存在。研究人员在实验中使用GAPGD和β -肌动蛋白进行基因表达正常化时应考虑这些组织学结果。
Housekeeping genes are widely used as internal controls for gene expression normalization for western blotting, northern blotting, RT-PCR, etc. They are generally thought to be expressed in all cells of the organism at similar levels because it is assumed that these genes are required for the maintenance of basic cellular function as constitutive genes. However, real-time RT-PCR experiments revealed that their expression may vary depending on the developmental stage, type of tissue examined, experimental condition, and so on. To date, no histological data on their expression are available for embryonic development. In the present study, we compared the histological expression profile of two commonly used housekeeping genes, GAPDH and beta-actin, in the developing chicken embryo by using section and whole mount in situ hybridization supplemented by RT-PCR. Our results show that neither GAPDH mRNA nor beta-actin mRNA is expressed in all cell types or tissues at high levels. Strikingly, expression levels are very low in some organs. Moreover, the two genes show partially complementary expression patterns in the liver, the vascular system and the digestive tract. For example, GAPDH is more strongly expressed in the liver than beta-actin, but at lower levels in the arteries. Vice versa, beta-actin is more strongly expressed in the gizzard than GAPDH, but it is almost absent from cardiac muscle cells. Researchers should consider these histological results when using GAPGD and beta-actin for gene expression normalization in their experiments.