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Blood, Vol. 106, Issue 7, 2580-2589, October 1, 2005
Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2)
Blood Galy et al.
106: 2580
Supplemental materials for: Galy et al, Vol 106, Issue 7, 2580-2589
Files in this Data Supplement:
- Figure S1. IronChip analysis of hepatic, duodenal, and splenic gene-expression profiles in Irp2–/– mice (JPG, 30.1 KB)
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Total RNA from the duodenum, liver, and spleen of IRP2-deficient mice was analyzed using the Mouse Version 3.0 of the IronChip microarray platform as described previously.1,2 This custom cDNA-based microarray covers approximately 300 genes involved in iron metabolism and interlinked biochemical pathways (eg, oxygen, copper). We compared mRNA expression levels in samples prepared from pools of four wild-type versus four Irp2–/– mice. A second experiment was carried out independently with distinct pools of mice (four wild-type versus four mutant mice, age and sex matched) and with a swap of the fluorescent dyes to avoid misinterpretations due to labeling artifacts3. The two arrays were analyzed separately. One of these two experiments is represented here. The wild-type sample was labeled with Cy3 (y axis); the Irp2–/– sample, with Cy5 (x axis). A spike in controls1 is represented by red dots; genes expressed in mouse tissues are shown in gray. The regression line and the 2-fold cut-off ratio line for regulation are represented in black and in red, respectively. Setting a cut-off value of 1.45-fold for regulation, we found a remarkable preservation of gene expression patterns in the duodenum, liver, and spleen of IRP2-deficient animals (raw data can be accessed at http://www.embl-heidelberg.de/ExternalInfo/hentze/suppinfo.html). The level of hydroxylmethylbilane synthase (Hmbs) mRNA (represented by the circled pink spots corresponding to 3 different ESTs, each spotted several times on the array) was found to be increased in pooled Irp2–/– spleens. However, analysis of Hmbs expression in individual animals by RNase protection assay revealed high interindividual variability and did not confirm statistically significant differences between mutant and wild-type mice (data not shown). These data show that the expression of IRP1 suffices to stabilize the cellular iron-regulatory network such that large changes in mRNA expression are avoided, and indicate a significant functional redundancy between IRP1 and IRP2. References 1. Richter A, Schwager C, Hentze S, Ansorge W, Hentze MW, Muckenthaler M. Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays. Biotechniques. 2002;33:620-630.
2. Muckenthaler M, Roy CN, Custodio AO, et al. Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet. 2003;34:102-107.
3. Benes V, and Muckenthaler M. Standardization of protocols in cDNA microarray analysis. Trends Biochem Sci. 2003;28:244-249.
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