Kang In Nee, Lee Chong Yew, Tan Suat Cheng
School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.
School of Pharmaceutical Sciences, Main Campus, Universiti Sains Malaysia, Penang, Malaysia.
Heliyon. 2019 Aug 1;5(7):e02156. doi: 10.1016/j.heliyon.2019.e02156. eCollection 2019 Jul.
Whilst the potential of neural stem cell (NSC)-based treatment is recognized worldwide and seems to offer a promising therapeutic option for stroke treatments, there is currently no full understanding regarding the effects of hypoxic and baicalein-enriched fraction (BEF) preconditioning approaches on the therapeutic potential of these cells for stroke. The potential of preconditioned NSC can be determined based on the expression of several key neuroprotective genes using qRT-PCR technique. However, prior to that, it is imperative and extremely important to carefully select reference gene(s) for accurate qRT-PCR data normalization to avoid error in data interpretation. This study aimed to evaluate the stability of ten candidate reference genes via comprehensive analysis using three algorithms software: geNorm, NormFinder and BestKeeper. Our results revealed that were the most reliable reference genes for BEF-preconditioned NSCs, but ironically, was ranked as the least stable reference gene for hypoxic-preconditioned NSCs. On the other hand, and were selected as the most stably expressed pair of reference genes for hypoxic-preconditioned NSCs. In conclusion, this study has pointed out the importance of identifying valid reference genes and has presented the first significant validation on best reference genes recommended for qRT-PCR study involves NSC preconditioned with hypoxia or with BEF extracted from medicinal plant.
虽然基于神经干细胞（NSC）的治疗潜力在全球范围内得到认可，并且似乎为中风治疗提供了一种有前景的治疗选择，但目前对于缺氧和富含黄芩苷组分（BEF）预处理方法对这些细胞治疗中风潜力的影响尚未完全了解。预处理的神经干细胞的潜力可以通过使用qRT-PCR技术检测几个关键神经保护基因的表达来确定。然而，在此之前，为了准确进行qRT-PCR数据归一化以避免数据解释错误，谨慎选择参考基因是至关重要且极其重要的。本研究旨在通过使用三种算法软件geNorm、NormFinder和BestKeeper进行综合分析，评估十个候选参考基因的稳定性。我们的结果表明，[具体基因名称1]是BEF预处理的神经干细胞最可靠的参考基因，但具有讽刺意味的是，[具体基因名称2]被列为缺氧预处理的神经干细胞最不稳定的参考基因。另一方面，[具体基因名称3]和[具体基因名称4]被选为缺氧预处理的神经干细胞最稳定表达的一对参考基因。总之，本研究指出了鉴定有效参考基因的重要性，并首次对推荐用于涉及缺氧预处理或用从[药用植物名称]提取的BEF预处理的神经干细胞的qRT-PCR研究的最佳参考基因进行了重要验证。
