Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

Journal of Animal Science and Biotechnology

Peter L Mente¹  Audrey T O’Nan²  Melissa S Ashwell²  Ryan S McCulloch¹ 

[1] University of North Carolina, Chapel Hill, North Carolina, USA;Animal Science Department, North Carolina State University, Raleigh, NC, USA

关键词: Stability;    Reference;    Porcine;    Normalization;    Housekeeping;    Cartilage;   

Others  :  813357
DOI  :  10.1186/2049-1891-3-36

 received in 2012-05-17, accepted in 2012-10-10,  发布年份 2012

【 摘 要 】

Background

Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.

Results

Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.

Conclusions

BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

【 授权许可】

   
2012 McCulloch et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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