Hif2α

HIF-1α, HIF-2α, and negative control siRNAs were purchased from Sigma. Primary astrocytes were transfected with the indicated combinations of siRNA against HIF-1α or/and HIF-2α at a final concentration of 10 nM or negative control siRNA at a final concentration of 10 nM using LipofectAmine RNAiMAX transfection reagent (Invitrogen), according to the manufacturer’s recommendation. At 24 or 48 h after transfection, cells were exposed to hypoxic conditions for 12 or 24 h.

Total RNA was extracted from liver, kidney and bone marrow using the PerfectPure RNA Tissue kit (Gentra Systems, Minneapolis, MN, USA) and cDNAs were synthesized using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real time PCR (qPCR) assays were performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) as previously described [15 (link),17 (link)], using the ABI Prism 7300 Sequence detection System (Applied Biosystems, Foster City, CA). Primers for quantification of Hepcidin, HIF-2 α, EPO, EPOR and β-actin (Sigma-Aldrich, Rechovot, Israel) are summarized in (Table 1). Each sample was analyzed in triplicate in individual assays. The specificity of the reaction is derived by the detection of the melting temperatures (Tms) of the amplification products immediately after the last reaction cycle. The target genes expression value was calculated by the ΔΔct method after normalization with a housekeeping gene (β-actin). Bone marrow aspirates cells were first suspended in RNA Save solution (Biological Industries, Beit Haemek, Israel). The remaining analysis was similar to the above mentioned methods.