Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids
Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids
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DOI：
10.1186/1472-6750-6-24
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发表时间：
2006-04-27
影响因子：
3.5
通讯作者：
Martens, Ann
中科院分区：
文献类型：
--
作者：
Bogaert, Lies;Van Poucke, Mario;Martens, Ann
背景资料：实时定量PCR是一种非常强大和准确的技术，可以在不同的生物条件下检测基因转录模式。比较转录谱的关键步骤之一是准确的标准化。在大多数发表的关于马的实时PCR的研究中，仅针对一个参考基因（通常为GAPDH或ACTB）进行标准化，而没有验证其表达稳定性。这可能导致不可靠的结论，因为已经证明，所谓的“管家基因”的表达水平在不同的组织、细胞类型或疾病阶段中，特别是在与恶性疾病相关的临床样品中可能有相当大的差异。本研究的目的是建立一套可靠的参考基因的研究有关正常马皮肤和马肉样瘤，这是最常见的皮肤tumor in horses.Results：在本研究中的基因转录水平的6个常用的参考基因（ACTB，B2 M，HPRT 1，UBB，TUBA 1和RPL 32）在正常马皮肤和马肉样瘤。应用geNorm小程序后，这组基因，TUBA1，ACTB和UBB被发现是最稳定的正常皮肤和B2M，ACTB和UBB在马sarcoid.Conclusion：基于这些结果，TUBA1，ACTB和UBB，分别B2M，ACTB和UBB可以被建议作为参考基因面板为正常马皮肤，分别马sarcoid.Conclusion定量数据的准确标准化。当比较正常皮肤和马类肉瘤时，建议使用UBB、ACTB和B2M的几何平均值作为可靠和准确的标准化因子。
Background: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses.Results: In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids.Conclusion: Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.