Open Access Research article

Genomic selection of reference genes for real-time PCR in human myocardium

Anna P Pilbrow1, Leigh J Ellmers1, Michael A Black2, Christine S Moravec3, Wendy E Sweet3, Richard W Troughton1, A Mark Richards1, Chris M Frampton1 and Vicky A Cameron1*

Author Affiliations

1 Christchurch Cardioendocrine Research Group, Department of Medicine, University of Otago-Christchurch, PO Box 4345, Christchurch 8014, New Zealand

2 Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand

3 Kaufman Center for Heart Failure, Department of Cardiovascular Medicine, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

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BMC Medical Genomics 2008, 1:64  doi:10.1186/1755-8794-1-64

Published: 29 December 2008

Abstract

Background

Reliability of real-time PCR (RT-qPCR) data is dependent on the use of appropriate reference gene(s) for normalization. To date, no validated reference genes have been reported for normalizing gene expression in human myocardium. This study aimed to identify validated reference genes for use in gene expression studies of failed and non-failed human myocardium.

Methods

Bioinformatic analysis of published human heart gene expression arrays (195 failed hearts, 16 donor hearts) was used to identify 10 stable and abundant genes for further testing. The expression stability of these genes was investigated in 28 failed and 28 non-failed human myocardium samples by RT-qPCR using geNorm software.

Results

Signal recognition particle 14 kDa (SRP14), tumor protein, translationally-controlled 1 (TPT1) and eukaryotic elongation factor 1A1 (EEF1A1) were ranked the most stable genes. The commonly used reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was ranked the least stable of the genes tested. The normalization strategy was tested by comparing RT-qPCR data of both normalized and raw expression levels of brain natriuretic peptide precursor (NPPB), a gene known to be up-regulated in heart failure. Non-normalized levels of NPPB exhibited a marginally significant difference between failed and non-failed samples (p = 0.058). In contrast, normalized NPPB expression levels were significantly higher in heart-failed patients compared with controls (p = 0.023).

Conclusion

This study used publicly available gene array data to identify a strategy for normalization involving two reference genes in combination that may have broad application for accurate and reliable normalization of RT-qPCR data in failed and non-failed human myocardium.