Agilent small rna kit

Whole blood (2.5 ml) was collected into PaXgene Blood RNA Tubes (PreAnalytix). The tubes were inverted 8–10 times then stored at room temperature for at least 2 hours. The tubes were frozen (−80 °C) and thawed overnight before RNA isolation (both miRNA and total RNA) with a PAXgene Blood microRNA Kit (Qiagen) including the DNase Set using the QiaCube. The concentrations and purity of the RNA samples were evaluated spectrophotometrically (BioPhotomer, Eppendorf). The RNA isolation process was validated by analyzing the integrity of several RNAs with the RNA 6000 Nano Chip Kit (Agilent). The presence of the small RNA fraction was confirmed by the Agilent Small RNA Kit (Agilent).

The RNA contained in extracellular vesicles/exosomes was isolated and purified using a phenol-free lysis buffer and rapid spin columns (SeraMir kit System Biosciences, Mountain View, CA). We performed RNA separation, detection and quantitation with the Agilent Small RNA Kit and a Bioanalyzer instrument (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA). The global microRNAs (miRs) profile was obtained using NanoString human microarrays (human V2 miRNA array >800 probes, Nanostring Technologies, Seattle, WA). To account for differences in hybridization and purification, data were normalized to the average counts for all control spikes in each sample using a proprietary bioinformatics software (nSolver™ Analysis Software 2.5, Nanostring Technologies, Seattle, WA). Briefly, we calculated a background level of expression for each sample using the mean level of the negative controls plus two standard deviations of the mean. MiRNA expressing less than two standard deviations from the mean were set to 0 expression. Those miRNAs that were considered non-zero expression, were normalized using a scaling factor based on the top 100 expressing miRNAs across all samples. For each sample, the average of the geometric means of the top 100 expressing miRNAs across all samples was divided by the geometric mean of each sample.[17 ]

Total RNA was extracted from 128 plasma samples using the QIAGEN miRNeasy serum/plasma kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer protocol. The miRNeasy Serum/Plasma Spike-In Control (lyophilized C. elegans miR-39 miRNA) was used to verify extraction quality and normalize TaqMan qPCR results. The RNA yield and purity were assessed using an Agilent Small RNA kit on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). In samples with low miRNA concentration (<100 pg/μl), the isolation procedure was repeated using the double initial plasma volume (400 µl).

Total cellular RNA, including miRNA, was isolated with the miRNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. All optional washing steps were included and RNA was eluted in a final volume of 30 μl RNase-free water. Total exosomal RNA, including miRNA, was isolated using the protocol described for cells with slight modifications: Exosome samples were pre-treated with 100 ng/μl RNAse A (Roche) for 30 min at 37°C, immediately before extracting RNA. Prior to the addition of chloroform and phase separation, 12 μg glycogen from Mytilus edulis (Sigma) were added to the sample. RNA concentrations were measured with the NanoDrop ND-1000 at 260 nm. RNA quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany) using the Agilent RNA 6000 Pico Kit (total RNA) and the Agilent Small RNA Kit (small RNA). The 2100 Bioanalyzer Expert Software B.02.08. (Agilent) was applied to generate electropherograms. RNA samples were stored at -80°C until further analysis.