Evaluation of candidate reference genes for quantitative real-time PCR analysis in a male rat model of dietary iron deficiency.
Evaluation of candidate reference genes for quantitative real-time PCR analysis in a male rat model of dietary iron deficiency.
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DOI：
10.1186/s12263-021-00698-0
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发表时间：
2021-10-02
影响因子：
3.5
通讯作者：
Clarke SL
中科院分区：
文献类型：
--
作者：
Fiddler JL;Clarke SL
实时定量聚合酶链反应（qPCR）是一种可靠而有效的基因表达定量方法。由于qPCR在检查营养与基因相互作用方面的使用越来越多，因此检查、开发和利用标准化方法进行数据分析和解释非常重要。用于标准化表达数据的常见方法涉及使用参考基因（RG）来确定相对mRNA丰度。在计算相对丰度时，RG的选择可能会影响实验结果，并有可能使数据解释发生偏差。虽然普通RG可用于标准化，但通常很少考虑RG选择对实验条件或各种组织或细胞类型之间的适用性。在当前的研究中，我们使用BestKeeper、比较δ定量循环、Normands和Refmands在从缺铁和成对喂养的铁充足大鼠获得的各种组织中检查了基因表达的稳定性，以确定在十个候选RG中的最佳选择。我们的研究结果表明，几种常用的RG（例如，Actb和Gapdh）与其他候选RG（例如，Rpl19和Rps29）。对于所有评估的RG，Tfrc的表达显着增加，在缺铁动物肝脏相比，铁充满配对喂养的控制，然而，相对诱导变化近4倍之间的最合适的（Rpl19）和最不合适的（Gapdh）RG。这些结果表明，RG的选择和使用应根据经验确定，RG选择可能会因实验条件和生物组织而异。
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.
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影响因子：
29
作者：
Anderson SA;Nizzi CP;Chang YI;Deck KM;Schmidt PJ;Galy B;Damnernsawad A;Broman AT;Kendziorski C;Hentze MW;Fleming MD;Zhang J;Eisenstein RS
通讯作者：
Eisenstein RS
影响因子：
3.7
作者：
de Jonge HJ;Fehrmann RS;de Bont ES;Hofstra RM;Gerbens F;Kamps WA;de Vries EG;van der Zee AG;te Meerman GJ;ter Elst A
通讯作者：
ter Elst A
影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
4.2
作者：
Erikson, KM;Pinero, DJ;Beard, JL
通讯作者：
Beard, JL
影响因子：
3.7
作者：
Najafpanah MJ;Sadeghi M;Bakhtiarizadeh MR
通讯作者：
Bakhtiarizadeh MR