Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs
Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs
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DOI：
10.1016/j.vetimm.2016.01.001
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发表时间：
2016-02-01
影响因子：
1.8
通讯作者：
Kaiser, P.
中科院分区：
文献类型：
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作者：
Borowska, D.;Rothwell, L.;Kaiser, P.
定量聚合酶链反应（qPCR）是用于定量基因表达，特别是参与免疫应答的基因的有力技术。虽然qPCR是一种非常有效和灵敏的工具，但酶促效率、RNA质量和抑制剂存在的变化可能导致错误。因此，需要对qPCR进行归一化，以获得可靠的结果并进行比较。最常见的方法是在qPCR分析中使用参考基因作为内部对照。在本研究中，七个基因，包括β-肌动蛋白（ACTB），β-2-微球蛋白（B2M），甘油醛-3-磷酸脱氢酶（GAPDH），β-葡萄糖醛酸酶（GUSB），TATA盒结合蛋白（TBP），α-微管蛋白（TUBAT）和28 S核糖体RNA（r28 S）的表达，从鸡淋巴组织中分离的细胞，并用三种不同的有丝分裂原刺激。使用geNorm、Normalware和BestKeeper软件测量基因的稳定性。来自geNorm和Normandy的结果是，该组中三个最稳定表达的基因是TBP、GAPDH和r28S。BestKeeper没有产生明确的答案，因为高度异质性的样本集。基于这些数据，我们将在未来的qPCR标准化中纳入TBP。该研究显示了在qPCR分析之前在其他组织中进行适当的参考基因标准化的重要性。(C)© 2016 Elsevier B.V.版权所有。
Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), TATA box binding protein (TBP), a-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. (C) 2016 Elsevier B.V. All rights reserved.