Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors.
Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs. Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan(®) assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms. Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes. For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended.
University Centre for Orthopaedics & Trauma Surgery and Centre for Translational Bone, Joint & Soft Tissue Research, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Germany. Electronic address: theresa.schildberg@uniklinikum-dresden.de.
Theresa
T
Schildberg
Juliane
J
Rauh
Henriette
H
Bretschneider
Maik
M
Stiehler
D000368:Aged; D000465:Algorithms; D001854:Bone Marrow Cells / Q000166:cytology*; D015870:Gene Expression; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D060888:Real-Time Polymerase Chain Reaction; D035181:TATA-Box Binding Protein / Q000235:genetics* / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics* / Q000378:metabolism
C459802:CASC3 protein, human; C107282:IPO8 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D035181:TATA-Box Binding Protein; D028961:beta Karyopherins