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PMID: 19114010 Published · epublish English Journal Article

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

BMC medical genomics ·Vol. 1 ·2008-12-29 ·Pages 64

Pilbrow AP, Ellmers LJ, Black MA, Moravec CS, Sweet WE, Troughton RW, Richards AM, Frampton CM, Cameron VA

Abstract

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. 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. 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). 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.

Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Pilbrow Anna P
Christchurch Cardioendocrine Research Group, Department of Medicine, University of Otago-Christchurch, PO Box 4345, Christchurch 8014, New Zealand. [email protected]
Ellmers Leigh J
Black Michael A
Moravec Christine S
Sweet Wendy E
Troughton Richard W
Richards A Mark
Frampton Chris M
Cameron Vicky A
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Article Info
Journal
BMC medical genomics
Abbr.
BMC Med Genomics
ISSN
1755-8794
DOI
10.1186/1755-8794-1-64
Email
[email protected]
Published
2008-12-29
Epub
2008-00-29
Pages
64
Language
English
Region
England
NLM ID
101319628
PMCID
PMC2632664
External Links
PubMed source DOI: 10.1186/1755-8794-1-64 PMC Full Text View journal details
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