Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

Dongrim Seol; Hyeonghun Choe; Hongjun Zheng; Keewoong Jang; Prem S Ramakrishnan; Tae-Hong Lim; James A Martin

doi:10.1186/1756-0500-4-162

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Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

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Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

Dongrim Seol, Hyeonghun Choe, Hongjun Zheng, Keewoong Jang, Prem S Ramakrishnan, Tae-Hong Lim and James A Martin

BMC research notes, Vol.4(1), pp.162-162

2011

DOI: 10.1186/1756-0500-4-162

PMCID: PMC3118343

PMID: 21615931

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Abstract

Background: 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. 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). Findings: In 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. Conclusions: Our 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.

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Title: Subtitle: Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc
Creators: Dongrim Seol - Department of Orthopedics and Rehabilitation, University of Iowa, 1182 ML, Iowa city, IA 52242, USA
Hyeonghun Choe - Department of Orthopedics and Rehabilitation, University of Iowa, 1182 ML, Iowa city, IA 52242, USA
Hongjun Zheng - Department of Orthopedics and Rehabilitation, University of Iowa, 1182 ML, Iowa city, IA 52242, USA
Keewoong Jang - Department of Orthopedics and Rehabilitation, University of Iowa, 1182 ML, Iowa city, IA 52242, USA
Prem S Ramakrishnan - Department of Orthopedics and Rehabilitation, University of Iowa, 1182 ML, Iowa city, IA 52242, USA
Tae-Hong Lim - Department of Biomedical Engineering, University of Iowa, 1402 SC, Iowa city, IA 52242, USA
James A Martin - Department of Orthopedics and Rehabilitation, University of Iowa, 1182 ML, Iowa city, IA 52242, USA
Resource Type: Journal article
Publication Details: BMC research notes, Vol.4(1), pp.162-162
DOI: 10.1186/1756-0500-4-162
PMID: 21615931
PMCID: PMC3118343
NLM abbreviation: BMC Res Notes
ISSN: 1756-0500
eISSN: 1756-0500
Publisher: BioMed Central
Language: English
Date published: 2011
Academic Unit: Roy J. Carver Department of Biomedical Engineering; Orthodontics; Pharmaceutical Sciences and Experimental Therapeutics; Orthopedics and Rehabilitation
Record Identifier: 9984040257202771

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