Department of Pharmacy, The First Affiliated Hospital of Shantou University Medical College, Shantou, 515041, Guangdong, China.
BMC Neurosci. 2018 Feb 1;19(1):3. doi: 10.1186/s12868-018-0403-6.
Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a critical tool for evaluating the levels of mRNA transcribed from genes. Reliable RT-qPCR results largely depend on normalization to suitable reference genes. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) are models that are commonly used to study ischemic stroke. However, the proper reference genes for RNA analysis in these two models have not yet been determined.
In this study, we evaluated the expression levels of six candidate housekeeping genes and selected the most suitable reference genes for RT-qPCR analyses of the cortices of MCAO mice and OGD/R-injured N2a cells. Four software programs, geNorm, NormFinder, BestKeeper and RefFinder, were used to validate the stabilities of the candidate reference genes. The results revealed that HPRT and 18S were the most stable reference genes in the cortices of MCAO mice and that β-actin and cyclophilin were the most stable reference genes in the OGD/R-injured N2a cells; in contrast, GAPDH and Sdha were the least stable genes in the cortices of MCAO mice and the OGD/R-injured N2a cells, respectively. Moreover, a combination of HPRT, 18S and cyclophilin was most suitable for normalization in analyses of the cortices of MCAO mice, and a combination of β-actin, cyclophilin, GAPDH, and 18S was most suitable for analyses of the OGD/R-injured N2a cells.
This study provides appropriate reference genes for further RT-qPCR analyses of in vivo and in vitro ischemic stroke and demonstrates the necessity of validating reference genes for RNA analyses under variable conditions.
实时逆转录定量聚合酶链反应（RT-qPCR）是评估从基因转录的 mRNA 水平的关键工具。可靠的 RT-qPCR 结果在很大程度上依赖于对合适的参考基因进行标准化。大脑中动脉闭塞（MCAO）和氧葡萄糖剥夺/再灌注（OGD/R）是常用于研究缺血性中风的模型。然而，这两种模型中 RNA 分析的合适参考基因尚未确定。
在这项研究中，我们评估了六个候选管家基因的表达水平，并选择了用于 MCAO 小鼠皮质和 OGD/R 损伤的 N2a 细胞 RT-qPCR 分析的最合适的参考基因。使用 geNorm、NormFinder、BestKeeper 和 RefFinder 这四个软件程序来验证候选参考基因的稳定性。结果表明，HPRT 和 18S 是 MCAO 小鼠皮质中最稳定的参考基因，β-肌动蛋白和环孢素是 OGD/R 损伤的 N2a 细胞中最稳定的参考基因；相比之下，GAPDH 和 Sdha 是 MCAO 小鼠皮质和 OGD/R 损伤的 N2a 细胞中最不稳定的基因。此外，HPRT、18S 和环孢素的组合最适合用于 MCAO 小鼠皮质分析的归一化，而β-肌动蛋白、环孢素、GAPDH 和 18S 的组合最适合用于 OGD/R 损伤的 N2a 细胞分析。
本研究为进一步进行体内和体外缺血性中风的 RT-qPCR 分析提供了合适的参考基因，并证明了在不同条件下验证 RNA 分析参考基因的必要性。
