Gene-expression profiles from iron-manipulated HeLa cells.

HeLa cells were treated with 100 μM hemin (H) or with 100 μM desferrioxamine (D) for 8 hours, and total RNA was purified from the cells. Fluorescent probes synthesized from total RNA derived from hemin-treated cells were labeled with Cy5-modified deoxyuridine 5-triphosphates (dUTPs), and those synthesized from total RNA derived from desferrioxamine-treated cells were labeled with Cy3-modified dUTPs; all were analyzed on the IronChip. (A) Virtual IronChip. Colors correspond to the calculated compensated ratios. Red spots represent genes with increased mRNA levels in hemin-treated cells. Green spots represent genes with increased mRNA levels in desferrioxamine-treated cells. Yellow spots represent genes that are equally expressed in both conditions tested. Selected genes are annotated. (B) Scatter plot analysis. Signals corresponding to the desferrioxamine-treated sample are represented on the y-axis. Signals corresponding to the hemin-treated sample are represented on the x-axis. In the experiment shown here, a gene is considered to be differentially expressed if the H/D ratio is calculated above 1.4 or below 0.7. Genes with a calculated H/D ratio above 1.4 (1.4-fold) are shown in red. Genes with a calculated H/D ratio below 0.7 (−1.4-fold) are represented in green. Housekeeping genes, like GAPDH and actin, are represented in yellow, and negative controls are shown in blue. Positive spike-in controls53 that have been added in equal amounts to the total RNA of hemin- and desferrioxamine-treated cells and thus by definition should not appear regulated are shown in pink. (C) Northern blot analysis of selected mRNAs. The ratios of signals obtained in H- and D-treated cells (as quantified on a Fluoroimager) are indicated.