Department of Animal Science, Michigan State University, East Lansing 48824, USA.
J Dairy Sci. 2011 Oct;94(10):4950-61. doi: 10.3168/jds.2011-4147.
Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set of genes that are not co-regulated must be analyzed to obtain accurate results. The objective of this study was to propose an alternative experimental and analytical protocol to assess the invariance of reference genes in porcine mammary tissue using mammary RNA and DNA concentrations as correction factors. Mammary glands were biopsied from 4 sows on d 110 of gestation (prepartum), on d 5 (early) and 17 (peak) of lactation, and on d 5 after weaning (postweaning). Relative expression of 7 potential reference genes, API5, MRPL39, VAPB, ACTB, GAPDH, RPS23, and MTG1, and one candidate gene, SLC7A1, was quantified by RT-qPCR using a relative standard curve approach. Variation in gene expression levels, measured as cycles to threshold at each stage of mammary physiological activity, was tested using a linear mixed model fitting RNA and DNA concentrations as covariates. Results were compared with those obtained with geNorm analysis, and genes selected by each method were used to normalize SLC7A1. Quantified relative mRNA abundance of GAPDH and MRPL39 remained unchanged across stages of mammary physiological activity after accounting for changes in tissue RNA and DNA concentration. In contrast, geNorm analysis selected MTG1, MRPL39, and VAPB as the best reference genes. However, when target gene SLC7A1 was normalized with genes selected either based on our proposed protocol or by geNorm, fold changes in mRNA abundance did not differ. In conclusion, the proposed analytical protocol assesses expression invariance of potential reference genes by accounting for variation in tissue RNA and DNA concentrations and thus represents an alternative method to select suitable reference genes for RT-qPCR analysis.
组织中细胞活性的变化会引起 RNA 浓度的变化，这会影响逆转录定量 PCR（RT-qPCR）分析中基因 mRNA 丰度的有效性。一种常见的方法是使用参考基因进行归一化。可以使用 geNorm 等程序选择合适的参考基因，尽管必须分析大量不协同调节的基因才能获得准确的结果。本研究的目的是提出一种替代的实验和分析方案，使用乳腺 RNA 和 DNA 浓度作为校正因子来评估猪乳腺组织中参考基因的不变性。在妊娠第 110 天（产前）、第 5 天（早期）和第 17 天（高峰期）以及断奶后第 5 天（断奶后），从 4 头母猪的乳腺组织中采集活检。使用相对标准曲线方法，通过 RT-qPCR 定量分析 7 个潜在参考基因（API5、MRPL39、VAPB、ACTB、GAPDH、RPS23 和 MTG1）和一个候选基因（SLC7A1）的相对表达。使用线性混合模型拟合 RNA 和 DNA 浓度作为协变量，测试基因表达水平的变化，以每个乳腺生理活动阶段的阈值循环表示。将结果与 geNorm 分析获得的结果进行比较，并使用每种方法选择的基因来归一化 SLC7A1。在考虑组织 RNA 和 DNA 浓度变化后，GAPDH 和 MRPL39 的相对 mRNA 丰度在乳腺生理活动的各个阶段保持不变。相比之下，geNorm 分析选择 MTG1、MRPL39 和 VAPB 作为最佳参考基因。然而，当用我们提出的方案或 geNorm 选择的基因来归一化靶基因 SLC7A1 时，mRNA 丰度的倍数变化没有差异。总之，所提出的分析方案通过考虑组织 RNA 和 DNA 浓度的变化来评估潜在参考基因表达的不变性，因此是选择 RT-qPCR 分析合适参考基因的替代方法。
