Department of Biochemistry, National University of Singapore, 119260, Singapore.
BMC Genomics. 2010 Jan 29;11:75. doi: 10.1186/1471-2164-11-75.
Gene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs), beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions.
Twenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder.
The ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes during neuronal differentiation of PC12 cells, regardless of the type of differentiation conditions. The combination of these two novel reference genes, but not the commonly used HKG, GAPDH, allows robust and accurate normalization of differentially expressed genes during PC12 differentiation.
转录水平的基因调控可以很好地反映生理和病理过程中复杂的信号机制。微阵列和实时定量 PCR 等转录组学方法需要稳定的参考基因来准确归一化基因表达。一些（但不是全部）研究表明，管家基因（HGK）β-肌动蛋白（ACTB）和甘油醛-3-磷酸脱氢酶（GAPDH），这些基因通常用于归一化，可能因细胞/组织类型和实验条件而异。目前尚不清楚这些基因在神经元分化过程中形态发生剧烈变化的细胞中是否稳定表达。最近对微阵列数据集的荟萃分析表明，一些（但不是全部）核糖体蛋白基因稳定表达。为了检验一些核糖体蛋白基因可作为神经元分化参考基因的假设，进行了全基因组分析，并根据表达稳定性确定了推定的参考基因。然后在六种分化条件下通过反转录实时定量 PCR 分析这些潜在参考基因的稳定性。
从 GDNF 和 NGF 诱导 PC12 细胞分化的基因表达谱微阵列分析中选择了 20 个稳定表达的基因，包括 13 个核糖体蛋白基因。然后，通过反转录实时定量 PCR 进一步分析了这些候选基因以及 ACTB 和 GAPDH 在包括 NGF、GDNF、forskolin、KCl 和 ROCK 抑制剂 Y27632 在内的多种刺激物诱导的 PC12 细胞分化中的表达水平。使用两种独立的统计方法，geNorm 和 NormFinder，评估了这些候选基因作为稳定参考基因的性能。
在 PC12 细胞的神经元分化过程中，鉴定出核糖体蛋白基因 RPL19 和 RPL29 是合适的参考基因，无论分化条件的类型如何。这两个新的参考基因的组合，而不是常用的 HGK GAPDH，允许在 PC12 分化过程中对差异表达基因进行稳健和准确的归一化。
