Comprehensive selection of reference genes for quantitative RT-PCR analysis of murine extramedullary hematopoiesis during development.
Comprehensive selection of reference genes for quantitative RT-PCR analysis of murine extramedullary hematopoiesis during development.
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DOI：
10.1371/journal.pone.0181881
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Gosain A
中科院分区：
文献类型：
--
作者：
Medrano G;Guan P;Barlow-Anacker AJ;Gosain A
本研究的目的是进行全面的评估和选择参考基因的研究髓外造血在发展过程中和出生后早期。通过实时定量PCR评估了在围产期五个时间点（胚胎第14.5、16.5、18.5天，出生后第0、21天）的四个器官（心脏、肝脏、脾脏和胸腺）中的总共六个候选参考基因（ACTB、GAPDH、HPRT 1、PPID、TBP、TUBB 3）。使用geNorm、NormFinder、Bestkeeper、Delta CT方法和RefFinder软件包分析候选参考基因的表达稳定性。详细的方法分离高质量/纯度的RNA和分析。详细的分析表明，TBP是胚胎样品的最佳单一参考基因，HPRT 1是出生后和合并的胚胎和出生后样品的最佳单一参考基因。器官水平分析表明HPRT 1是心脏、肝脏和胸腺样本的最佳参考基因，而TBP是脾脏样本的最佳候选基因。一般来说，TUBB 3始终是最不稳定的基因正常化。这是第一个研究描述了一个系统的全面选择的参考基因的小鼠髓外造血组织在一个发展的时间过程。我们提供了建议的参考基因的个别组织和发育阶段，并建议参考基因的组合提供了灵活性的实验设计和分析。
The purpose of this study was to perform a comprehensive evaluation and selection of reference genes for the study of extramedullary hematopoiesis during development and the early post-natal period. A total of six candidate reference genes (ACTB, GAPDH, HPRT1, PPID, TBP, TUBB3) in four organs (heart, liver, spleen, and thymus) over five perinatal time points (Embryonic days 14.5, 16.5, 18.5, Post-natal days 0, 21) were evaluated by quantitative real-time PCR. The expression stability of the candidate reference genes were analyzed using geNorm, NormFinder, Bestkeeper, Delta CT method, and RefFinder software packages. Detailed methodology for isolation of high quality/purity RNA and analysis is presented. Detailed analysis demonstrated that TBP is the best single reference gene for embryonic samples and HPRT1 is the best single reference gene for post-natal and pooled embryonic and post-natal samples. Organ-level analysis demonstrated that HPRT1 was the most suitable reference gene for heart, liver and thymus samples, while TBP was the best candidate for spleen samples. In general, TUBB3 was consistently the least stable gene for normalization. This is the first study to describe a systematic comprehensive selection of reference genes for murine extramedullary hematopoietic tissues over a developmental time course. We provide suggested reference genes for individual tissues and developmental stages and propose that a combination of reference genes affords flexibility in experimental design and analysis.