Evaluation of reference genes for human chondrocytes cultured in several different thermal environments
Evaluation of reference genes for human chondrocytes cultured in several different thermal environments
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DOI：
10.3109/02656736.2014.906048
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发表时间：
2014-01-01
影响因子：
3.1
通讯作者：
Kuroki, Hiroshi
中科院分区：
文献类型：
--
作者：
Ito, Akira;Aoyama, Tomoki;Kuroki, Hiroshi
目的：本研究的目的是鉴定在几种不同热环境和不同培养系统中培养的软骨细胞中显示稳定表达的内参基因。材料和方法：将人关节软骨细胞采用单层或颗粒培养系统在32℃、37℃和41℃下培养3天。然后，提取总RNA，进行定量实时逆转录聚合酶链式反应(qRT-PCR)。使用三种不同的算法（geNorm、NormFinder 和 BestKeeper）对 qRT-PCR 数据进行分析，从 7 个候选内参基因（B2M、ACTB、GAPDH、HSPCB、RPL13a、YWHAZ 和 18S）中鉴定出表现出稳定表达的内参基因。结果：除 HSPCB 和 YWHAZ 外，候选内参基因在表达上表现出系统性变异。在单层培养中，RPL13a 是使用 NormFinder 和 BestKeeper 鉴定出的最稳定的基因；在使用 geNorm 时，ACTB 和 GAPDH 显示出最高的表达稳定性。在沉淀培养中，ACTB 是使用 NormFinder 和 BestKeeper 确定的最稳定的基因，而 GAPDH 和 RPL13a 是使用 geNorm 确定的最稳定的参考基因。在组合组中，B2M 和 GAPDH 是使用 geNorm 鉴定的最稳定的基因，而 RPL13a 和 YWHAZ 分别是根据 NormFinder 和 BestKeeper 鉴定的最稳定的基因。使用 NormFinder 确定的所有组中两个候选内参基因的最佳组合是 RPL13a 和 YWHAZ。 结论：RPL13a 和 YWHAZ 的组合可能适合作为在 32°C、37°C 和 41°C 单层、颗粒或组合培养中培养的人软骨细胞的内参基因。
Purpose: The purpose of this study was to identify reference genes showing stable expression in chondrocytes cultured under several different thermal environments and in different culture systems.Materials and methods: Human articular chondrocytes were cultured by monolayer or pellet culture system at 32 degrees C, 37 degrees C, and 41 degrees C for 3 days. Thereafter, the total RNA was extracted, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed. The qRT-PCR data was analysed using three different algorithms (geNorm, NormFinder, and BestKeeper) to identify reference genes exhibiting stable expression from among the seven candidate reference genes (B2M, ACTB, GAPDH, HSPCB, RPL13a, YWHAZ, and 18S).Results: The candidate reference genes, except for HSPCB and YWHAZ, showed systematic variations in expression. In the monolayer culture, RPL13a was the most stable gene identified using NormFinder and BestKeeper; on using geNorm, ACTB and GAPDH showed the highest expression stability. In the pellet culture, ACTB was the most stable gene identified using NormFinder and BestKeeper, whereas GAPDH and RPL13a were the most stable reference genes as determined using geNorm. In the combined group, B2M and GAPDH were the most stable genes identified using geNorm, whereas RPL13a and YWHAZ were the most stable as per NormFinder and BestKeeper, respectively. The best combination of two candidate reference genes among all the groups determined using NormFinder was RPL13a and YWHAZ.Conclusion: The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32 degrees C, 37 degrees C, and 41 degrees C in monolayer, pellet, or combined cultures.