Fig. 7
qPCR analysis to validate significant microglial transcripts identified by translatome profiling. A cDNAs synthesized from the purified RNA after immunoprecipitation of cortical lysates from vehicle or ABX-treated WT-CD11br and vehicle or ABX-treated APPPS1-21-CD11br male and female mice (n = 3 per group) were used for qPCR analysis. APPPS1-21 male and female mice were used as controls for pull-down assay. Gapdh was used as reference gene. The relative expression levels of microglia transcripts Hexb (a, bonafide microglia marker), Sirpa (b, immune response), Calm1, Camk2n1 (c-d, Calcium Signaling), Bdh1(e, Ketone metabolism), Atp8 (f, Mitochondrial ATP synthase) and Atp6v0d1(g, lysosomal v-ATPase) are determined using qPCR by the ΔΔCT method and plotted as a bar graph normalized to the expression of WT-vehicle treated male mice. The error bar indicates the SD of two technical replicates. UD refers to undetermined. B Hexb, Calm1, Atp8 and Atp6v0d1 transcripts were chosen for further validation using cDNA prepared from RNA purified from FACS-isolated microglia. APPPS1-21 male and female mice treated with vehicle or ABX were sacrificed at 7-weeks. 3-hemicortices from each group were pooled. Following Percoll-density centrifugation, Flow-cytometry was used to isolate CD11bhi + CD45 lo cells (enriched in microglia). The cells were immediately used for RNA isolation using Trizol reagent. The relative expression of Hexb (a), Calm1(b), Atp8 (c) and Atp6v0d1 (d) was determined by ΔΔCT method and plotted as a bar graph normalized to the expression of vehicle-treated male AD (APPPS1-21) mice. The error bar represents the SD of two experimental replicates. Statistical significance of the effect of ABX-treatment and sex on expression of the transcripts were indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns-not significant, based on Tukey’s Multiple comparison test. ns indicates no significance