Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, School of Food Science and Engineering, Inner Mongolia Agricultural University, Huhhot, China.
J Ind Microbiol Biotechnol. 2011 Sep;38(9):1279-86. doi: 10.1007/s10295-010-0906-3. Epub 2010 Nov 23.
Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.
干酪乳杆菌 Zhang 是从中国内蒙古自制马奶酒中分离出来的一种潜在益生菌菌株，已被测序并保存在 GenBank 中。实时定量 PCR 是研究干酪乳杆菌 Zhang 相关基因表达水平最广泛使用的方法之一。为了进行准确可靠的基因表达分析，使用一个或多个合适的内参基因对基因表达数据进行标准化是必不可少的。我们使用了三种统计方法（geNorm、NormFinder 和 BestKeeper）来评估五个候选内参基因（GAPD、gyrB、LDH、16s rRNA 和 recA）在不同培养条件和不同生长阶段下的表达水平，以找到一个合适的管家基因，可作为内部标准。结果表明，最佳的参考基因是 GAPD，在所有测试的不同实验条件下，建议将 GAPD 和 gyrB（最稳定的参考基因）这两个基因作为实时定量 PCR 实验的归一化基因。对候选内参基因的系统验证对于获得干酪乳杆菌 Zhang 基因表达水平的实时定量 PCR 研究可靠的分析结果非常重要。
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