Fiddler Joanna L, Clarke Stephen L
Division of Nutritional Sciences, Cornell University, Ithaca, NY, 14850-6301, USA.
Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK, 74078, USA.
Genes Nutr. 2021 Oct 2;16(1):17. doi: 10.1186/s12263-021-00698-0.
Quantitative real-time polymerase chain reaction (qPCR) is a reliable and efficient method for quantitation of gene expression. Due to the increased use of qPCR in examining nutrient-gene interactions, it is important to examine, develop, and utilize standardized approaches for data analyses and interpretation. A common method used to normalize expression data involves the use of reference genes (RG) to determine relative mRNA abundance. When calculating the relative abundance, the selection of RG can influence experimental results and has the potential to skew data interpretation. Although common RG may be used for normalization, often little consideration is given to the suitability of RG selection for an experimental condition or between various tissue or cell types. In the current study, we examined the stability of gene expression using BestKeeper, comparative delta quantitation cycle, NormFinder, and RefFinder in a variety of tissues obtained from iron-deficient and pair-fed iron-replete rats to determine the optimal selection among ten candidate RG.
Our results suggest that several commonly used RG (e.g., Actb and Gapdh) exhibit less stability compared to other candidate RG (e.g., Rpl19 and Rps29) in both iron-deficient and iron-replete pair-fed conditions. For all evaluated RG, Tfrc expression significantly increased in iron-deficient animal livers compared to the iron-replete pair-fed controls; however, the relative induction varied nearly 4-fold between the most suitable (Rpl19) and least suitable (Gapdh) RG.
These results indicate the selection and use of RG should be empirically determined and RG selection may vary across experimental conditions and biological tissues.
定量实时聚合酶链反应（qPCR）是一种可靠且高效的基因表达定量方法。由于qPCR在研究营养物质与基因相互作用中的应用日益增加，因此研究、开发和采用标准化的数据分析与解释方法非常重要。一种常用于标准化表达数据的方法是使用参考基因（RG）来确定相对mRNA丰度。在计算相对丰度时，RG的选择会影响实验结果，并可能导致数据解释出现偏差。尽管常用的RG可用于标准化，但通常很少考虑RG选择对于实验条件或不同组织或细胞类型的适用性。在本研究中，我们使用BestKeeper、比较△定量循环、NormFinder和RefFinder在从缺铁和配对喂养的铁充足大鼠获得的各种组织中检测基因表达的稳定性，以确定十个候选RG中的最佳选择。
我们的结果表明，在缺铁和铁充足的配对喂养条件下，与其他候选RG（例如Rpl19和Rps29）相比，几种常用的RG（例如Actb和Gapdh）表现出较低的稳定性。对于所有评估的RG，与铁充足的配对喂养对照组相比，缺铁动物肝脏中的Tfrc表达显著增加；然而，在最合适的（Rpl19）和最不合适的（Gapdh）RG之间，相对诱导变化近4倍。
这些结果表明，RG的选择和使用应根据经验确定，并且RG的选择可能因实验条件和生物组织而异。
