Abstract
Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.
Highlights
ATDC5 cells, which represent progenitor cells for chondroblasts, could form cell aggregates when growing in a medium supplemented with insulin and exhibit the entire spectrum of endochondral bone development [1,2]
We aimed to find out the suitable reference genes for quantitative reverse transcription-polymerase chain reaction (qPCR) analysis during chondrocyte differentiation of ATDC5 cells
Stability analysis of 16 reference genes To determine stable reference genes for in vitro chondrogenesis of ATDC5 cells, cells cultured in growth medium (GM), insulin medium (IM) and CM were analyzed at 1, 3, 5, 7 and 14 d
Summary
ATDC5 cells, which represent progenitor cells for chondroblasts, could form cell aggregates when growing in a medium supplemented with insulin and exhibit the entire spectrum of endochondral bone development [1,2]. Suitable reference genes are employed to normalize cell number, RNA extraction and reverse transcription efficiency differences. Single reference gene, such as GAPDH, Actb and 18 s rRNA, has been generally used for normalization in more than 90% of studies [7,8]. Our previous study on chondrogenesis of ATDC5 cells found that the data was always in divergence or even in contradiction when using GAPDH and Actb as reference genes One or both of these two housekeeping genes was unsuitable for qPCR normalization under ATDC5 chondrocyte differentiation condition. The selection of suitable housekeeping gene for specific study is a prerequisite for qPCR assay to obtain reliable data
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