Total RNA was isolated using Trizol Reagent (Life Technologies, CA, USA) according to the manufacturer’s procedure. RNA yields and A260 ratios were determined by spectrophotometry. The RNA isolation was followed by DNase treatment (TURBO DNA-free DNase Treatment & Removal Reagents, Ambion, CA, USA) for the removal of genomic DNA. Purified total RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). For further analysis, only samples with RNA integrity numbers (RIN) ranging from 6.4 to 8.3 were used.
Each sample was reverse transcribed using 0.75 µg of purified total RNA, a mixture of oligo(dT) and random hexamer primers, and RNase H + iScript reverse transcriptase (iScript cDNA synthesis kit, Bio-Rad, CA, USA). Transcript relative quantifications were performed on a LightCycler 480 II Instrument (Roche, IN, USA) in 384-well plates. Each real-time quantitative PCR reaction was run in triplicate. Primer sequences are listed in Supplementary Table 2. GAPDH, ACTB, and PGK1 were used as housekeeping genes. The fold difference in expression of the genes of interest between non-demented controls (n = 25) and AD Braak I-V cases (n = 49) was calculated by the 2−∆∆Ct method [19 (link)].
Each sample was reverse transcribed using 0.75 µg of purified total RNA, a mixture of oligo(dT) and random hexamer primers, and RNase H + iScript reverse transcriptase (iScript cDNA synthesis kit, Bio-Rad, CA, USA). Transcript relative quantifications were performed on a LightCycler 480 II Instrument (Roche, IN, USA) in 384-well plates. Each real-time quantitative PCR reaction was run in triplicate. Primer sequences are listed in Supplementary Table 2. GAPDH, ACTB, and PGK1 were used as housekeeping genes. The fold difference in expression of the genes of interest between non-demented controls (n = 25) and AD Braak I-V cases (n = 49) was calculated by the 2−∆∆Ct method [19 (link)].