The examples of 4 methods used to confirm ATRA-modulated genes in the differentiation of NB4 cells.
(A) A partial view of a cDNA array result by comparing the transcriptional expression pattern in untreated NB4 cells (left) with that in ATRA-treated NB4 cells (right). The signals of housekeeping genes appeared in the bottom as an internal control. (B) The results of semiquantitative RT-PCR. The up-regulation of CD52 and the down-regulation of myeloperoxidase seem to be protein synthesis–independent, while the up-regulation of transglutaminase and myeloid progenitor inhibitory factor was completely suppressed by the protein synthesis inhibitor cycloheximide. In the figure, A denotes ATRA; C, cycloheximide; and Ac, a combination of ATRA and cycloheximide. The number denotes hours after treatment. (C) The results of Northern blotting, with the lower panel showing the loading control (28s rRNA). (D) Relative mRNA levels of 3 novel genes (RIG-K, RIG-C, and RIG-I) were measured by semiquantitative RT-PCR and the Taq-man real-time quantitative PCR assay using the same samples. Data from theTaq-man analysis were normalized to mRNA concentrations according to the concentration of internal control actin and plotted relative to the level at time zero. The expression profiles of these genes, as measured by these 2 independent methods, were quite similar.
