title: Optimization of Reference Genes for Normalization of Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction Results in Senescence Study of Mesenchymal Stem Cells.
abstract: Recently, it has been suggested that cellular senescence is associated with stem cell exhaustion, which reduces the regenerative potential of tissues and contributes to aging and age-related diseases. Mesenchymal stem cells (MSCs) attract a large amount of attention in stem cell research and regeneration medicine because they possess multiple advantages and senescent MSCs could be one of the most useful stem cell models in aging studies. It is important to quantitatively evaluate senescence markers to both identify and study the mechanisms involved in MSC senescence. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the most widely used tool to quantify the mRNA levels of markers. However, no report has demonstrated the optimal reference genes that should be used to normalize RT-qPCR in senescence studies of MSCs. In this study, we compared 16 commonly used reference genes (GAPDH, ACTB, RPL13A, TBP, B2M, GUSB, RPLPO, YWHAZ, RPS18, EEF1A1, ATP5F1, HPRT1, PGK1, TFRC, UBC, and PPIA) in proliferating or replicative-senescent human adipose-derived MSCs (hAD-MSCs) that were isolated from seven healthy donors aged 29-59 years old. Three algorithms (geNorm, NormFinder, and BestKeeper) were used to determine the most optimal reference gene. The results showed that PPIA exhibited the most stable expression during senescence, while the widely used ACTB exhibited the lowest stability. We also confirmed that different reference genes lead to different evaluations of senescence markers. Our work ensures that results obtained from senescence studies of hAD-MSCs will be appropriately evaluated in both basic research and clinical trials.
authors.affiliation: 1 Institute of Basic Medical Sciences, School of Basic Medicine, Peking Union Medical College, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences , Beijing, People's Republic of China .
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Su
authors.affiliation: 1 Institute of Basic Medical Sciences, School of Basic Medicine, Peking Union Medical College, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences , Beijing, People's Republic of China .|2 State Key Laboratory of Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences , Beijing, People's Republic of China .
authors.forename: Xinglei
authors.initials: X
authors.lastname: Yao
authors.affiliation: 3 Department of Oncology, School of Basic Medicine, Peking Union Medical College, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences , Beijing, People's Republic of China .
authors.forename: Zhao
authors.initials: Z
authors.lastname: Sun
authors.affiliation: 1 Institute of Basic Medical Sciences, School of Basic Medicine, Peking Union Medical College, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences , Beijing, People's Republic of China .
authors.forename: Qin
authors.initials: Q
authors.lastname: Han
authors.affiliation: 1 Institute of Basic Medical Sciences, School of Basic Medicine, Peking Union Medical College, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences , Beijing, People's Republic of China .
authors.forename: Robert Chunhua
authors.initials: RC
authors.lastname: Zhao
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D000328:Adult; D015153:Blotting, Western; D016922:Cellular Senescence / Q000235:genetics*; D017931:DNA Primers / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005796:Genes; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D008875:Middle Aged; D008954:Models, Biological; D012313:RNA / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D012984:Software
chemical_list: D017931:DNA Primers; D012313:RNA