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首页> 外文期刊>BMC Medical Genomics >Genomic selection of reference genes for real-time PCR in human myocardium
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Genomic selection of reference genes for real-time PCR in human myocardium

机译：人心肌实时PCR参考基因的基因组选择

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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.
机译：背景技术实时PCR（RT-qPCR）数据的可靠性取决于使用适当的参考基因进行标准化。迄今为止，还没有关于使人心肌中的基因表达正常化的有效参考基因的报道。这项研究旨在鉴定经过验证的参考基因，用于失败和未失败的人心肌的基因表达研究。方法使用已发表的人类心脏基因表达阵列（195个衰竭心脏，16个供体心脏）的生物信息学分析来鉴定10个稳定和丰富的基因，以进行进一步测试。通过使用geNorm软件的RT-qPCR，在28个失败的和28个未失败的人心肌样本中研究了这些基因的表达稳定性。结果信号识别颗粒14 kDa（SRP14），肿瘤蛋白，翻译控制1（TPT1）和真核延伸因子1A1（EEF1A1）被评为最稳定的基因。常用的参考基因甘油醛-3-磷酸脱氢酶（GAPDH）在测试的基因中排名最不稳定。通过比较脑钠肽前体（NPPB）的标准化表达水平和原始表达水平的RT-qPCR数据，测试了标准化策略。 NPPB的未归一化水平在失败和未失败的样本之间显示出微不足道的差异（p = 0.058）。相反，与对照组相比，心衰患者的正常NPPB表达水平显着更高（p = 0.023）。结论本研究使用可公开获得的基因阵列数据来确定涉及两个参考基因的标准化策略，这些策略可能广泛应用于对失败和未失败的人心肌进行RT-qPCR数据的准确可靠的标准化。

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