Pérez Silvia, Royo Luis J, Astudillo Aurora, Escudero Dolores, Alvarez Francisco, Rodríguez Aida, Gómez Enrique, Otero Jesús
Unidad de Coordinación de Trasplantes y Terapia Celular, Hospital Universitario Central de Asturias, C/Celestino Villamil s/n, 33006 Oviedo, Spain.
BMC Mol Biol. 2007 Dec 20;8:114. doi: 10.1186/1471-2199-8-114.
Quantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue.
Our findings indicate that out of ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT), PPIA, RPLP and GADPH show the most stable gene transcription levels in left ventricle specimens obtained from organ donors, as assessed using geNorm and Normfinder software. The expression of TBP was found to be highly regulated.
We propose the use of PPIA, RPLP and GADPH as reference genes for the accurate normalisation of qRT-PCR performed on heart tissue. TBP should not be used as a control in this type of tissue.
定量实时逆转录PCR（qRT-PCR）是评估不同组织中基因表达的有用工具，但选择合适的对照对于标准化结果至关重要，从而避免差异并最大化灵敏度和准确性。到目前为止，许多基因已被用作单一参考基因，而此前并未验证它们作为对照的价值。这种做法可能导致错误的结论，最近的证据表明需要使用来自几个对照基因数据的几何平均值。在此，我们确定了一组合适的基因，用作定量人类心脏组织中基因表达的内参。
我们的研究结果表明，在十个常用的参考基因（GADPH、PPIA、ACTB、YWHAZ、RRN18S、B2M、UBC、TBP、RPLP和HPRT）中，使用geNorm和Normfinder软件评估发现，PPIA、RPLP和GADPH在从器官捐赠者获得的左心室标本中显示出最稳定的基因转录水平。发现TBP的表达受到高度调控。
我们建议使用PPIA、RPLP和GADPH作为参考基因，用于对心脏组织进行的qRT-PCR的准确标准化。TBP不应在这类组织中用作对照。
