Ribosomal Protein L13a as a Reference Gene for Human Bone Marrow-Derived Mesenchymal Stromal Cells During Expansion, Adipo-, Chondro-, and Osteogenesis
Ribosomal Protein L13a as a Reference Gene for Human Bone Marrow-Derived Mesenchymal Stromal Cells During Expansion, Adipo-, Chondro-, and Osteogenesis
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DOI：
10.1089/ten.tec.2012.0081
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发表时间：
2012-10-01
影响因子：
3
通讯作者：
Maniura-Weber, Katharina
中科院分区：
文献类型：
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作者：
Studer, Deborah;Lischer, Stefanie;Maniura-Weber, Katharina
在人间充质基质细胞 (MSC) 研究领域，定量实时逆转录聚合酶链式反应 (qPCR) 是研究分化、施加刺激或抑制剂或 siRNA 等因素时基因表达模式变化的首选方法。为了可靠地检测微小变化，使用在所有条件下稳定表达的参考基因 (RG) 至关重要。现场使用的大量不同的 RG 以及缺乏对其适用性的验证使得研究之间的比较变得不可能。因此，这项工作旨在建立一种单一的 RG，用于使用 MSC 进行中胚层分化研究。七种常用的 RG（3-磷酸甘油醛脱氢酶 [GAPDH]、核糖体蛋白 L13a [RPL13a]、β-肌动蛋白 [ACTB]、酪氨酸 3-单加氧酶/色氨酸 5-单加氧酶激活蛋白、zeta 多肽 [YWHAZ]、真核翻译延伸因子 1 α [EF1 α]、β研究了 2-微球蛋白 [B2M] 和 18S 核糖体 RNA [18S]）在骨髓源性 MSC 扩增至四代期间以及诱导后第 9、16 和 22 天的脂肪、软骨和成骨分化期间的 mRNA 表达稳定性。 RPL13a 经验证可用于 MSC（骨髓和胎盘来源）的 qPCR 研究，此外还适用于原代人骨细胞 (HBC) 和骨肉瘤细胞系 MG-63。 GAPDH 和 ACTB 是两种最常用的 RG，表现出最高的表达方差。 RPL13a 的卓越性能应使其成为所有解决中胚层分化问题的 MSC 研究的首选 RG。
In the field of human mesenchymal stromal cell (MSC) research, quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the method of choice to study changes in gene expression patterns upon differentiation, application of stimuli, or of factors such as inhibitors or siRNAs. To reliably detect small changes, the use of a reference gene (RG) that is stably expressed under all conditions is essential. The large number of different RGs used in the field and the lack of validation of their suitability make the comparison between studies impossible. Therefore, this work aims to establish one single RG for mesodermal differentiation studies that use MSCs. Seven commonly used RGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal protein L13a [RPL13a], beta-actin [ACTB], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide [YWHAZ], eukaryotic translational elongation factor 1 alpha [EF1 alpha], beta 2-microglobulin [B2M], and 18S ribosomal RNA [18S]) were investigated concerning their mRNA expression stability during expansion of bone marrow-derived MSCs up to four passages as well as during their adipo-, chondro-, and osteogenenic differentiation on days 9, 16, and 22 after induction. RPL13a was validated for qPCR studies of MSCs (bone marrow-and placenta-derived) and, additionally, for primary human bone cells (HBCs) and the osteosarcoma cell line MG-63. GAPDH and ACTB, the two most frequently used RGs, showed the highest expression variance. The superior performance of RPL13a should make it the RG of choice for all MSC studies addressing mesodermal differentiation.