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. 2018 Jan 22;14(1):e1006802. doi: 10.1371/journal.ppat.1006802

Fig 2. Regional and subtype-dependent miRNA expression confirmation and representative miRNA incorporation into functional complexes.

Fig 2

(A) Validation of the small RNA-Seq signature in selected miRNAs by RT-qPCR analysis in the FC (upper panel) and CB (lower panel) of controls, sCJD MM1 and VV2. Results were normalized to the housekeeping genes U6 snRNA (figure) and RNU5 expression, which showed similar results in the expression analysis of deregulated miRNAs between control and sCJD cases. Housekeeping gene levels remained unaltered between groups. Normalization was performed relative to controls. Error bars indicate SD. (B) Detection of sCJD-related miRNAs by RT-qPCR in Ago immunoprecipitates from the FC of controls (n = 3) and sCJD MM1 brain homogenates (n = 3). The two Ago antibodies used (11A9 and H-300) were able to immunoprecipitate Ago-2 from brain tissue (left panel) and reported similar results in the specific enrichment of sCJD-related miRNAs (146a-5p, 26a-5p, 195-5p, 154-5p, 204-5p and 5701) in the FC of sCJD MM1 cases (right panels). miRNA-204-5p was selected as negative control, since no changes were detected between control and sCJD cases with small RNA-Seq and RT-qPCR analysis. Non-specific immunoglobulins (NS) were used as control antibody for the immunoprecipitation. Error bars indicate SD. (C) Upper panel: western blot analysis of Ago-2 (11A9 antibody) in the FC and CB control, sCJD MM1 and VV2 cases. Four representative cases per diagnostic group and brain region are shown in the western blot. Quantifications derived from densitometry analysis were performed in 15 cases per diagnostic group (n = 15/group). β-actin was used as loading control. Densitometries of the western blots are shown. Normalization was performed relative to controls. Error bars indicate SD. Bottom panel: Gene expression levels of Ago-2 in the FC and CB of controls, sCJD MM1 and VV2 cases by RT-qPCR. Results were normalized to housekeeping genes GAPDH (figure) and GUSB with similar results. Housekeeping levels remained unaltered between groups. 10 cases per diagnostic group (n = 10/group) were analyzed. In all cases, statistical significance (compared to controls) was set at *p<0.05.