Department of Medicine, University of Adelaide, Adelaide, S.A., Australia.
Chemotherapy. 2011;57(1):43-53. doi: 10.1159/000321477. Epub 2011 Feb 1.
BACKGROUND/AIMS: Mucositis is the term used to describe damage caused by chemotherapy to mucous membranes of the alimentary tract. RT-PCR has recently been utilised to determine the molecular events that occur in mucositis. As this method relies on the use of a validated endogenous control, this study aims to validate commonly used housekeeping genes in an irinotecan-induced mucositis model.
Rats were administered irinotecan and sacrificed at different time points, in particular 1, 24, 72 and 144 h following treatment. Histopathological damage was assessed by haematoxylin and eosin staining. RT-PCR was used to evaluate the expression of 11 housekeeping genes. Expression stability was determined by the Normfinder program. Matrix metalloproteinase 2 was used as a target gene to validate the appropriateness of the top-ranking housekeeping gene.
For normalisation to multiple housekeeping genes, the most stable combination across all time points in the jejunum was Ywhaz/UBC and in the colon UBC/β-actin. SDHA and GAPDH were the most variable genes in the jejunum and colon where they were 4.4 and 3.2 fold upregulated following irinotecan, respectively.
For normalisation of irinotecan-induced mucositis gene expression studies, a combination of Ywhaz/UBC and UBC/β-actin should be used in the jejunum and colon, respectively. UBC is the most favourable if restricted to a single housekeeping gene across all time points.
背景/目的：黏膜炎是指化疗对消化道黏膜造成的损伤。逆转录聚合酶链反应（RT-PCR）最近被用于确定黏膜炎中发生的分子事件。由于该方法依赖于使用经过验证的内参基因，因此本研究旨在在伊立替康诱导的黏膜炎模型中验证常用的管家基因。
大鼠给予伊立替康，并在不同时间点处死，特别是在治疗后 1、24、72 和 144 小时。苏木精-伊红染色评估组织病理学损伤。RT-PCR 用于评估 11 个管家基因的表达。通过 Normfinder 程序确定表达稳定性。基质金属蛋白酶 2 被用作靶基因来验证排名最高的管家基因的适宜性。
对于多个管家基因的归一化，空肠中所有时间点最稳定的组合是 Ywhaz/UBC，结肠中最稳定的组合是 UBC/β-actin。SDHA 和 GAPDH 是空肠和结肠中最易变的基因，在伊立替康处理后分别上调了 4.4 倍和 3.2 倍。
对于伊立替康诱导的黏膜炎基因表达研究的归一化，空肠和结肠中分别应使用 Ywhaz/UBC 和 UBC/β-actin 的组合。如果限制在单个管家基因，则 UBC 是最有利的。
