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Journal of Animal Science and Biotechnology

, 3:36

Animal Genetics

Abstract

BackgroundExpression 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.

ResultsTen 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.

ConclusionsBestKeeper, 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.

KeywordsCartilage Housekeeping Normalization Porcine Reference Stability AbbreviationsactbBeta actin

b2mBeta-2-microglobulin

cycle thresholdCt

gapdhGlyceraldehyde-3-phophate dehydrogenase

hmbsHydroxymethylbilane synthase

hprtHypoxanthine guanine phosphoribosyl transferase

osteoarthritisOA

ppiaPeptidylprolyl isomerase A

quantitative real-time polymerase chain reactionqPCR

rpl13aRibosomal protein L13a

s18Ribosomal protein S18

sdhaSuccinate dehydrogenase flavoprotein subunit A

tbpTATA box binding protein

ywhazTyrosine 3-monooxygenase-tryptophan 5-monooxygenase activation protein—zeta polypeptide.

Electronic supplementary materialThe online version of this article doi:10.1186-2049-1891-3-36 contains supplementary material, which is available to authorized users.

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Autor: Ryan S McCulloch - Melissa S Ashwell - Audrey T O’Nan - Peter L Mente

Fuente: https://link.springer.com/







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