Abstract
BackgroundThe accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD).FindingsIn vitro, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture.ConclusionsOur results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.
Highlights
The accuracy of quantitative real-time RT-PCR is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes
Our results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit intervertebral disc (IVD) culture studies
On the other hand, Pgk1 (1.02) and CycA (1.04) were ranked as second and third genes, respectively. This implies that Hprt1, CycA, and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in rat and Hprt1, Pgk1, and CycA are more potential reference genes compared to the others
Summary
The accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. The relevance and magnitude of absolute measures obtained from qRT-PCR are subject to inherent sample variations that usually lead to statistical uncertainty. Such outcomes may lead to inferences that may be biologically obscure [2]. Numerous studies on molecular level changes in disc biology have been reported by normalization using GAPDH [9,10,11,12] and Actb [13,14,15] without validation for their stability. The assumption of certain genes being constitutively expressed may be valid in certain cases, this assumption cannot be taken for granted under rapidly changing conditions such as growth, remodeling and disease
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