INRA, UMR1213 Herbivores, F-63122 Saint-Genès-Champanelle, France.
Animal. 2013 Aug;7(8):1344-53. doi: 10.1017/S1751731113000475. Epub 2013 Apr 4.
The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants.
逆转录定量实时 PCR（RT-qPCR）的可靠性取决于使用精心选择的稳定参考基因对 mRNA 丰度进行归一化。我们的目的是为牛或山羊脂肪组织（AT）、乳腺、肝脏和肌肉中的参考基因正常化提出一套参考基因。所有这些组织都有助于营养分配和代谢，从而影响反刍动物生产的盈利能力（即胴体、肉和奶）。在这项研究中，我们使用 geNorm 程序分析了属于不同功能类别的 8 个常用参考基因（CLN3、EIF3K、MRPL39、PPIA、RPLP0、TBP、TOP2B 和 UXT），以确定牛和/或山羊组织中最稳定的参考基因。参考基因的丰度和排名在组织、物种以及组织和/或物种的组合之间存在差异。因此，我们提出了 29 套参考基因，这些参考基因因组织和/或物种而异。例如，EIF3K、TOP2B 和 UXT 被提议为牛 AT 中最稳定的参考基因；UXT、EIF3K 和 RPLP0 是牛和山羊 AT 中最稳定的参考基因。在 29 组建议的参考基因中，有 27 组的数据归一化需要 3 个最佳参考基因。在其中两组中，当研究组织数量增加时，需要 4 到 5 个参考基因进行数据归一化。例如，UXT、EIF3K、TBP、TOP2B 和 CLN3 是牛乳腺、AT、肌肉和肝脏中数据归一化所需的参考基因。我们已经评估了一些我们建议的参考基因集，用于 CD36 基因表达的归一化。使用三个最稳定的参考基因进行归一化表明，以向日葵油为补充的浓缩物日粮下调了牛乳腺中 CD36 基因的表达，而日粮中包含快速降解淀粉则上调了山羊肝脏中 CD36 基因的表达。用最不稳定的参考基因进行归一化会消除 CD36 基因表达的膳食调节，这可能导致对 CD36 基因调节的错误解释。总之，我们的研究结果为其他旨在测量反刍动物组织和/或物种特异性 mRNA 丰度的研究提供了有价值的参考基因集。
