Microarray identification of functional modules involving Haptoglobin and/or Hemopexin

Haptoglobin and Hemopexin are plasma acute phase proteins that bind with high affinity hemoglobin and heme, respectively. Several studies have demonstrated that they have a key role in the protection against oxidative stress and inflammation. However, little is known about the functional modules in which they are involved. To gain insights into this issue, we integrated bioinformatic and experimental approaches to identify genes coexpressed with Haptoglobin or Hemopexin and modulated in Haptoglobin and/or Hemopexin knock-out mice. These genes have a high probability to be functionally related to Haptoglobin and/or Hemopexin. We performed a gene expression analysis of the livers of Hp- and/or Hx-null mice compared to wild-type controls. The mice used in the following experiments, i.e. wild-type (Hp+/Hx+/), Hp-null (Hp-/-Hx+/), Hx-null (Hp+/Hx-/-), and HpHx-null (Hp-/-Hx-/-), were littermates derived by breeding F1 double heterozygous Hp+/-Hx+/- mice in the mixed genetic background C57/BLJ6 X 129Sv. We have three experimental points: Hx-null mice, Hp-null mice and HpHx-null mice. At least 5 adult mice per genotype were perfused via aorta with PBS, sacrificed and their liver pooled. Thirty microgram of total RNA were subjected to direct labeling reaction by incorporation of cyanin 3 (Cy3) or cyanin 5 (Cy5) fluorescent dyes into the cDNA by priming with oligo(dT). Four replicates were set up for each experimental point. In order to exclude artifacts resulting from different dye usage, we employed the dye-swap approach.