Ren Shengwei, Zhang Feng, Li Changyou, Jia Changkai, Li Siyuan, Xi Haijie, Zhang Hongbo, Yang Lingling, Wang Yiqiang
Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, China.
Mol Vis. 2010 Jun 11;16:1076-86.
To evaluate the suitability of common housekeeping genes (HKGs) for use in quantitative reverse transcription PCR (qRT-PCR) assays of the cornea in various murine disease models.
CORNEAL DISEASE MODELS STUDIED WERE: 1) corneal neovascularization (CorNV) induced by suture or chemical burn, 2) corneal infection with Candida albicans or Aspergillus fumigatus by intrastromal injection of live spores, and 3) perforating corneal injury (PCI) in Balb/c mice or C57BL/6 mice. Expression of 8 HKGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin [ACTB], lactate dehydrogenase A [LDHA], ribosomal protein L5 [RPL5], ubiquitin C [UBC], peptidylprolyl isomerase A [PPIA], TATA-box binding protein [TBP1], and hypoxanthine guanine phosphoribosyl transferase [HPRT1]) in the cornea were measured at various time points by microarray hybridization or qRT-PCR and the data analyzed using geNorm and NormFinder.
Microarray results showed that under the CorNV condition the expression stability of the 8 HKGs decreased in order of PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA. qRT-PCR analyses demonstrated that expression of none of the 8 HKGs remained stable under all conditions, while GAPDH and ACTB were among the least stably expressed markers under most conditions. Both geNorm and NormFinder analyses proposed best HKGs or HKG combinations that differ between the various models. NormFinder proposed PPIA as best HKG for three CorNV models and PCI model, as well as UBC for two fungal keratitis models. geNorm analysis demonstrated that a similar model in different mice strains or caused by different stimuli may require different HKGs or HKG pairs for the best normalization. Namely, geNorm proposed PPIA and HRPT1 and PPIA and RPL5 pairs for chemical burn-induced CorNV in Balb/c and C57BL/6 mice, respectively, while UBC and HPRT1 and UBC and LDHA were best for Candida and Aspergillus induced keratitis in Balb/c mice, respectively.
When qRT-PCR is designed for studies of gene expression in murine cornea, preselection of situation-specific reference genes is recommended. In the absence of knowledge about situation-specific HKGs, PPIA and UBC, either alone or in combination with HPRT1 or RPL5, can be employed.
评估常用管家基因（HKGs）在各种小鼠疾病模型角膜定量逆转录聚合酶链反应（qRT-PCR）检测中的适用性。
所研究的角膜疾病模型包括：1）缝线或化学烧伤诱导的角膜新生血管形成（CorNV）；2）通过基质内注射活孢子进行白色念珠菌或烟曲霉角膜感染；3）Balb/c小鼠或C57BL/6小鼠的角膜穿孔伤（PCI）。通过微阵列杂交或qRT-PCR在不同时间点测量角膜中8种管家基因（甘油醛-3-磷酸脱氢酶[GAPDH]、β-肌动蛋白[ACTB]、乳酸脱氢酶A[LDHA]、核糖体蛋白L5[RPL5]、泛素C[UBC]、肽基脯氨酰异构酶A[PPIA]、TATA盒结合蛋白[TBP1]和次黄嘌呤鸟嘌呤磷酸核糖基转移酶[HPRT1]）的表达，并使用geNorm和NormFinder分析数据。
微阵列结果显示，在CorNV条件下，8种管家基因的表达稳定性按PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA的顺序降低。qRT-PCR分析表明，8种管家基因在所有条件下均未保持稳定表达，而GAPDH和ACTB在大多数条件下是表达最不稳定的标记物之一。geNorm和NormFinder分析均提出了不同模型中最佳的管家基因或管家基因组合。NormFinder提出PPIA是三种CorNV模型和PCI模型的最佳管家基因，而UBC是两种真菌性角膜炎模型的最佳管家基因。geNorm分析表明，不同小鼠品系或由不同刺激引起的相似模型可能需要不同的管家基因或管家基因对来进行最佳标准化。具体而言，geNorm分别提出PPIA与HRPT1组合以及PPIA与RPL5组合用于Balb/c和C57BL/6小鼠化学烧伤诱导的CorNV，而UBC与HPRT1组合以及UBC与LDHA组合分别是Balb/c小鼠白色念珠菌和烟曲霉诱导的角膜炎的最佳组合。
当设计qRT-PCR用于小鼠角膜基因表达研究时，建议预先选择适合具体情况的内参基因。在不了解具体情况的管家基因时，可以单独使用PPIA和UBC，或与HPRT1或RPL5联合使用。
