Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells.
Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells.
复制标题
验证管家基因作为白消安损伤微血管内皮细胞逆转录 qPCR 分析的参考
DOI：
10.1155/2018/4953806
复制
发表时间：
2018
影响因子：
--
通讯作者：
Zeng L
中科院分区：
文献类型：
--
作者：
Ju W;Smith AO;Sun T;Zhao P;Jiang Y;Liu L;Zhang T;Qi K;Qiao J;Xu K;Zeng L
内皮细胞(ECs)可以表达一些重要的细胞因子和信号分子，这些分子在正常的造血和再分化中起关键作用。白花丹诱导的血管内皮损伤是造血干细胞移植(HSCT)后的重要特征。但损伤内皮细胞如何影响造血重建的分子机制尚不清楚。这可能是通过调控某些基因表达的变化来实现的。RT-qPCR是从看家基因中选择稳定的参考基因(RG)来准确确定基因表达水平的最流行的方法之一。因此，我们的目的是选择稳定的RGS来更准确地测量白花丹诱导的血管内皮损伤过程中的mRNA水平。本研究选取14个RGS，研究其在白头翁处理内皮细胞损伤后72小时内的表达稳定性。我们的结果显示，与五种统计算法相比，RG的稳定性存在极大的差异。根据最终排名，ywhaz和alas1被认为是最空闲的两个RG，而最常用的两个RG(GAPDH和ACTB)不是最稳定的RG。接下来，通过检测基于上述四个基因YWHA、ALAS1、GAPDH和ACTB的信号通路基因CTNNB1、ROBO4和NOTCH1来验证这些数据。结果表明，使用不稳定的RGS对mRNA表达数据进行归一化处理对基因折叠变化有很大影响，这意味着生物学结论的可靠性值得怀疑。基于所使用的最佳RGS，我们还发现，在白丹受损的内皮细胞中，ROBO4显著过度表达。总之，我们的数据重申了RGS选择对于有效分析白花丹损伤的内皮细胞基因表达的重要性。这也为环磷酰胺、氟达拉滨等不同药物损伤内皮细胞更准确的差异表达基因筛选和进一步扩大生物分子研究提供了非常有用的指导和依据。
Endothelial cells (ECs) could express some important cytokines and signal molecules which play a key role in normal hematopoiesis and repopulation. Busulfan-induced vascular endothelial injury is an important feature after hematopoietic stem cell transplantation (HSCT). But the molecular mechanism of how the injured ECs affect hematopoietic reconstruction is still unknown. It is possibly through modulation of the change of some gene expression. RT-qPCR is one of the most popular methods used to accurately determine gene expression levels, based on stable reference gene (RG) selection from housekeeping genes. So our aim is to select stable RGs for more accurate measures of mRNA levels during Busulfan-induced vascular endothelial injury. In this study, 14 RGs were selected to investigate their expression stability in ECs during 72 hours of EC injury treated with Busulfan. Our results revealed extreme variation in RG stability compared by five statistical algorithms. ywhaz and alas1 were recognized as the two idlest RGs on account of the final ranking, while the two most usually used RGs (gapdh and actb) were not the most stable RGs. Next, these data were verified by testing signalling pathway genes ctnnb1, robo4, and notch1 based on the above four genes ywha, alas1, gapdh, and actb. It shows that the normalization of mRNA expression data using unstable RGs greatly affects gene fold change, which means the reliability of the biological conclusions is questionable. Based on the best RGs used, we also found that robo4 is significantly overexpressed in Busulfan-impaired ECs. In conclusion, our data reaffirms the importance of RGs selection for the valid analysis of gene expression in Busulfan-impaired ECs. And it also provides very useful guidance and basis for more accurate differential expression gene screening and future expanding biomolecule study of different drugs such as cyclophosphamide and fludarabine-injured ECs.
登录
查看更多内容
影响因子：
23.9
作者：
Butler JM;Nolan DJ;Vertes EL;Varnum-Finney B;Kobayashi H;Hooper AT;Seandel M;Shido K;White IA;Kobayashi M;Witte L;May C;Shawber C;Kimura Y;Kitajewski J;Rosenwaks Z;Bernstein ID;Rafii S
通讯作者：
Rafii S
影响因子：
2.7
作者：
Li, Xiuying;Yang, Qiwei;Bai, Jinping;Xuan, Yali;Wang, Yimin
通讯作者：
Wang, Yimin
影响因子：
4.6
作者：
Lv P;Ito T;Oogushi A;Nakashima K;Nagao M;Hibino T
通讯作者：
Hibino T
DOI：
10.1113/jp273350
发表时间：
2017-03-15
期刊：
The Journal of physiology
影响因子：
--
作者：
Patel R;Moffatt JD;Mourmoura E;Demaison L;Seed PT;Poston L;Tribe RM
通讯作者：
Tribe RM
同种异体移植后急性移植物抗宿主病患者骨髓内皮祖细胞的损伤
影响因子：
6.5
作者：
Cao, Xie-Na;Kong, Yuan;Huang, Xiao-Jun
通讯作者：
Huang, Xiao-Jun