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Validation of suitable reference genes for quantitative polymerase chain reaction analysis in rabbit bone marrow mesenchymal stem cell differentiation

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Bone marrow mesenchymal stem cells (BMSCs) are considered as multipotent cells, representing a multi­lineage potential to differentiate into mesodermal lineages of mesenchymal tissues, including cartilage, bone, fat, muscle and tendon. Tissue engineering in BMSCs has made great advances in the regeneration of cartilage and bone defects. To uncover the mechanisms of the multipotent differentiation process, the molecular changes in gene expression profiles during chondrogenic and osteogenic differentiation need to be evaluated with reliable, accurate, fast and sensitive methods. Reverse transcriptionquantitative polymerase chain reaction is a commonly used technology for analyzing gene expression, depending on an appropriate reference gene to normalize the errors. The commonly used reference genes vary, and no ideal and universal reference genes suitable for all conditions exist; therefore validation of the stability of gene expression is required. In the present study, three common statistical algorithms, geNorm, Normfinder and BestKeeper, were used to identify the expression stability of 12 genes, and the target differentiation markers during the differentiation of BMSCs were evaluated accurately. Our results demonstrated that YWHAZ, PPIA and GAPDH were suitable as reference genes for chondrogenic differentiation, while RPL13a allowed an efficient normalization expression value of interest genes for osteogenic differentiation of BMSCs. By contrast, the most unstable reference genes were 18s rRNA, B2M and HPRT1 in all studies, and these should be avoided when investigating the differentiation of BMSCs. Our results demonstrate validation of the appropriate reference genes for accurate gene expression in chondrogenic and osteogenic differentiation of BMSCs.
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Document Type: Research Article

Affiliations: 1: Department of Orthopedics, The First Hospital of Jilin University, Jilin University, Changchun, Jilin 130021, P.R. China 2: Central Laboratory, Jilin University, ChinaJapan Union Hospital, Changchun, Jilin 130033, P.R. China

Publication date: August 1, 2015

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  • Molecular Medicine Reports is a monthly, peer-reviewed journal available in print and online, that includes studies devoted to molecular medicine, underscoring aspects including pharmacology, pathology, genetics, neurosciences, infectious diseases, molecular cardiology and molecular surgery. In vitro and in vivo studies of experimental model systems pertaining to the mechanisms of a variety of diseases offer researchers the necessary tools and knowledge with which to aid the diagnosis and treatment of human diseases.
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