Institute of Life Sciences, Key Laboratory of Organ Development and Regeneration of Zhejiang Province, College of Life and Environment Sciences, Hangzhou Normal University, Zhejiang, 311121, China.
College of Life Sciences, Zhejiang University, Hangzhou, China.
Dev Neurobiol. 2018 Jan;78(1):39-50. doi: 10.1002/dneu.22558. Epub 2017 Nov 24.
Accurate quantification of gene expression is fundamental for understanding the molecular, genetic and functional bases of tissue development and diseases. Quantitative real-time PCR (qPCR) is now the most widely used method of quantifying gene expression due to its simplicity, specificity, sensitivity, and wide quantification range. The use of appropriate reference genes to ensure accurate normalization is crucial for the correct quantification of gene expression from the early development, maturation, aging to injury processes in the central nervous system (CNS). In this study, we have determined the expression profiles of 12 candidate housekeeping genes (ACTB, CYC1, HMBS, GAPDH, HPRT1, RPL13A, YWHAZ, PPIA, RPLP0, TFRC, GUS, and 18S rRNA) in developing mouse brain and spinal cord. Throughout development, there was a significant degree of fluctuations in their expression levels, indicating the importance and complexity of finding appropriate reference genes. Three software including BestKeeper, geNorm and NormFinder were used to evaluate the stability of potential reference genes. GUS was the most stable gene and GUS/YWHAZ were the most stable reference gene pair across different developmental stages in different CNS regions, whereas HPRT1 and GAPDH were the most variable genes and thus inappropriate to use as reference genes. Therefore, our results identified GUS and YWHAZ as the best combination of two reference genes for expression data normalization in CNS developmental studies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 39-50, 2018.
准确量化基因表达是理解组织发育和疾病的分子、遗传和功能基础的基础。由于其简单性、特异性、灵敏度和广泛的定量范围，实时定量 PCR（qPCR）现在是最广泛使用的定量基因表达的方法。由于正确定量中枢神经系统（CNS）从早期发育、成熟、衰老到损伤过程中的基因表达，使用适当的参考基因来确保准确的归一化至关重要。在这项研究中，我们确定了 12 个候选管家基因（ACTB、CYC1、HMBS、GAPDH、HPRT1、RPL13A、YWHAZ、PPIA、RPLP0、TFRC、GUS 和 18S rRNA）在发育中的小鼠大脑和脊髓中的表达谱。在整个发育过程中，它们的表达水平有很大程度的波动，这表明找到合适的参考基因的重要性和复杂性。包括 BestKeeper、geNorm 和 NormFinder 在内的三种软件用于评估潜在参考基因的稳定性。GUS 是最稳定的基因，GUS/YWHAZ 是不同 CNS 区域不同发育阶段最稳定的参考基因对，而 HPRT1 和 GAPDH 是最易变的基因，因此不适合用作参考基因。因此，我们的结果确定 GUS 和 YWHAZ 是 CNS 发育研究中表达数据归一化的最佳参考基因对。
