Abstract
BackgroundReliability 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.MethodsBioinformatic 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.ResultsSignal 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).ConclusionThis 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.
Highlights
Reliability of real-time PCR (RT-qPCR) data is dependent on the use of appropriate reference gene(s) for normalization
We have identified two genes, Signal recognition particle 14 kDa (SRP14) and TPT1, that in combination can be used for accurate and reliable normalization of RT-qPCR gene expression data within failed human myocardium and non-failed human myocardium separately, and two genes, TPT1 and eukaryotic elongation factor 1A1 (EEF1A1) that are more stably expressed than glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and may be used for normalization of RTqPCR data when comparing gene expression levels between failed and non-failed human myocardium samples
Our analysis indicated that the top five genes would be required for accurate normalization of RT-qPCR data within failed and non-failed human myocardium, the number of genes used needs to be a balance between accuracy and practical considerations
Summary
Reliability of real-time PCR (RT-qPCR) data is dependent on the use of appropriate reference gene(s) for normalization. Quantitative real-time PCR (RT-qPCR) is one of the most sensitive, specific, and widely-used methods for measuring the expression levels of selected genes [2,3]. Among the most commonly used reference genes in RTqPCR are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin [6] These genes were introduced for normalization when mRNA expression was assessed using semi-quantitative methods, such as Northern blotting and RNase protection, because of their relatively high abundance in numerous cell-types [1,4]. No validated universal or human heart-specific reference genes have been identified
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