Pcp cy3

The SurePrint G3 Human miRNA 8x60K Microarray (G4872A, Agilent Technologies) was used according to the manufacturer's protocols. Total RNA was isolated from GSCs with TRIzol reagent (Life Technologies). In total, 100 ng of total RNA was labelled with pCp-Cy3 (Agilent Technologies) and 15 units of T4 RNA ligase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 16 °C for 2 h. Labelled samples were purified with MicroBio-Spin six columns (Bio-Rad, Richmond, CA, USA) and hybridized to microarrays at 55 °C with rotation at 20 rpm for 20 h. The arrays were scanned using an Agilent Microarray Scanner (G2565BA, Agilent Technologies). The scanned images were analysed using the Feature Extraction software, version 10.7.3.1 (Agilent Technologies) with background correction. Data analysis was performed with GeneSpring GX, version 12.6.0 (Silicon Genetics). Expression data were normalized to the 75th percentile using the GeneSpring normalisation option with no substantial difference in results. The miRNA microarray analysis was performed in duplicate for each cell line. Statistical comparisons were made with the use of both the GeneSpring analysis-of-variance tool and volcano plot filtering.

Hippocampi of the right hemisphere were dissected from the hippocampus of one month-old AD11 and control mice and total RNA was isolated using Trizol (Invitrogen, San Diego, CA) and DNAse treated by Qiagen columns. Quality and integrity of each sample was controlled for using the Agilent BioAnalyzer 2100 (Agilent RNA 6000 nano kit). Only samples with a RNA Integrity Number (RIN) index higher than 8.0 were selected. All the experimental steps involving the labeling, hybridization and washings of the samples were done following the Agilent protocol (http://chem.agilent.com (accessed on 22 February 2021), miRNA Microarray System with miRNA Complete Labeling and Hyb Kit protocol, version 3.1.1, Agilent Technologies, Santa Clara, CA, USA). The Small RNA kit was used for the separation and quantification of miRNA. Labeled miRNAs were obtained from 100 ng of total RNA through the ligation of a 5′-cytidine bisphosphate-Cy3 (pCp-Cy3, Agilent Technologies) group at the 3′-end of each miRNA. We had previously treated total RNA with alkaline phosphatase (TaKaRa Bio Inc, Kusatsu, Japan) at 37 °C for 30 min. to enhance the T4 RNA-ligase (Ambion, ThermoFisher, Waltham, MA, USA) efficiency.