![]() ![]() In many instances, however, indirect measures are needed to further understand iron homeostasis. To investigate the iron content of biological tissues, inductively coupled plasma mass spectrometry is a useful strategy due to its low detection limits. Ī variety of biomarkers and methodologies exist to investigate iron status for example, measuring serum ferritin and transferrin saturation are common practices and often employed together to enhance the detection of systemic ID. Due to its importance in biological functions, inadequate levels of iron lead to microcytic anemia, diminished cognitive development, and decreased ATP production. In humans, iron deficiency (ID) remains the most common single nutrient deficiency and affects approximately 25% of the world’s population or 1.62 billion people according to the World Health Organization. Iron is an essential nutrient and is involved in many mammalian processes including DNA synthesis, erythropoiesis, ATP production, and oxygen transport. These results indicate the selection and use of RG should be empirically determined and RG selection may vary across experimental conditions and biological tissues. ![]() 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. 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. 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. 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. When calculating the relative abundance, the selection of RG can influence experimental results and has the potential to skew data interpretation. A common method used to normalize expression data involves the use of reference genes (RG) to determine relative mRNA abundance. 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. Quantitative real-time polymerase chain reaction (qPCR) is a reliable and efficient method for quantitation of gene expression.
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