title: Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.
abstract: 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.
authors.affiliation: Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, School of Food Science and Engineering, Inner Mongolia Agricultural University, Huhhot, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Zhao
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Pengfei
authors.initials: P
authors.lastname: Gao
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Sun
authors.forename: Tiansong
authors.initials: T
authors.lastname: Sun
authors.forename: Heping
authors.initials: H
authors.lastname: Zhang
mesh_terms: D002681:China; D015870:Gene Expression; D020869:Gene Expression Profiling; D005798:Genes, Bacterial; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000378:metabolism; D019936:Probiotics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards