Quantitative Analysis of Amyloid Transcripts in Mouse Brain

Brains of wild-type and APPPS1 mice previously treated for Tregs depletion or amplification were harvested at 4 months of age after transcardiac perfusion with PBS. Cerebellum was removed and total RNA was extracted from hemi-brains using RNeasy lipid tissue midi kit (Qiagen). Following DNase treatment, RNA quality was verified using an Agilent Bioanalyzer and quantity measured with a Nanodrop 1000 (ThermoFisher Scientific). For cDNA synthesis, 2 µg of total RNA were processed using RT² First Strand kit (Qiagen). Quality control of RNA and cDNA samples was assessed using RT² RNA QC PCR Array (Qiagen). Relative expression of specific mRNAs was assessed by SYBR green-based real-time quantitative PCR using RT² SYBR Green ROX (Qiagen) and LightCycler 96 Instrument (Roche). Specificity of all primers was validated using Primer BLAST database analysis, and PCR efficiency (> 90%) was validated after optimizing the concentration and annealing temperature. Amplification conditions were: 95 °C for 5 min, then 45 cycles at 95 °C for 10 s, 15 s at either 60 °C or 56 °C depending on the primer pairs, and 72 °C for 30 s. A melting curve was generated at the end of amplification cycles for assessing the specificity of the reaction. Hypoxanthine-guanine phosphoribosyltransférase (HPRT) and peptidylprolyl isomerase A (PPIA) were used as reference housekeeping genes for normalization. Relative expression of genes was evaluated as fold changes using the mean of the control group as reference, and was calculated as 2^−ΔΔCt. All primers were ordered from Eurogentec (sequences available in Additional file 8: Table S1). Data were analyzed using LightCycler 96 software (Roche) and the R software environment.