Approach for defining endogenous reference genes in gene expression experiments
Section snippets
Endothelial cells
HUVEC were isolated from five healthy donors by a modification of the method of Jaffe et al. [12]. After delivery, umbilical cords were conserved in cord buffer (4 mM KCl, 140 mM NaCl, 11 mM Hepes, 12 mM d-glucose, and 100 U/mL penicillin–streptomycin; Biowhittaker, USA) for up to 6 days at 4 °C. Briefly, cords were canulated in both ends and washed first with warm cord buffer and second with warm M199 (Biowhittaker) supplemented with 100 U/mL penicillin–streptomycin. Then, 10 mL of a filtered
Results
The quantification of the expression of the 10 selected genes was assayed by SYBR-green-based real-time PCR in all the samples. In the case of group A, human umbilical vein endothelial cells were subjected to different stimuli (TNFα, IL-4, IFNγ) for either 2, 6, or 18 h. Group B corresponded either to healthy individuals or to IgA nephropathy patients. The analysis of the raw data (Ct values) was performed in 10 cycles (Scheme 1). In each cycle, one of the selected genes was set as the potential
Discussion
Normalization is as important in gene expression studies as the techniques used to obtain the data. It allows researchers to compare expression profiles obtained in different laboratories or under different conditions. Although is clear that the use of an endogenous reference, often referred to as internal control genes, is the gold standard, there is little agreement on the conditions that a gene has to meet to be considered an “endogenous reference gene.” Nevertheless, it is clear that it
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