title: Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.
abstract: Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.
authors.affiliation: The Central Laboratory, China-Japan Union Hospital, Jilin University, 126 Xiantai Street, Changchun, 130033, Jilin, China.
authors.forename: Xiuying
authors.initials: X
authors.lastname: Li
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Yang
authors.forename: Jinping
authors.initials: J
authors.lastname: Bai
authors.forename: Yali
authors.initials: Y
authors.lastname: Xuan
authors.forename: Yimin
authors.initials: Y
authors.lastname: Wang
mesh_terms: D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D005796:Genes / Q000235:genetics; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D011506:Proteins