Ribogreen rna quantification

Animals were euthanized with a fatal overdose of sodium pentobarbital at P14 and immediately decapitated per IACUC approved protocol. The hippocampus was isolated, flash-frozen in liquid nitrogen, and stored at −80°C until further use. Total hippocampal RNA was isolated from one lobe using the RNAqueous RNA Isolation kit (Ambion; Austin, TX) and quantified by Nanodrop P1000 (Thermo Scientific; Waltham, MA). For RNAseq analysis, 4 mice per experimental group were studied. The sample size was determined based upon previously described power calculations to optimize detection of differentially-expressed genes.^{19 (link),20 (link)} RNA sequencing was performed by the University of Minnesota Genomics Center after quality check by RiboGreen RNA quantification (Invitrogen; Carlsbad, CA), and an Agilent 2100 Bioanalyzer (Agilent; Santa Clara, CA; Supplemental Table 1). RNA samples with RIN values ≥ 9.2 were used for library construction and sequenced at 50b paired-end (PE) at >25M reads/sample. For Real-time PCR validation, RNA was isolated from hippocampi in the same way as above (n=6/group) from a separate set of experimental mice. Selected mRNAs were quantified using Taqman gene expression probes (Supplemental Table 2) on a ThermoFisher QuantStudio 3 Real-Time PCR system. Tbp, which was not affected by iron deficiency,^{10 (link)} was used as a control gene.