Total RNA including miRNAs was isolated using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. In detail, 100µl serum was mixed with 100µl RNase-free water and 1ml Qiazol lysis solution and incubated for 5 min at room temperature. After addition of 200 µl chloroform, the samples were vigorously shaken for 15s and phase and centrifuged at 12.000 rpm and 4°C for 15 min. Aqueous phase was transferred into an empty tube and mixed with 3µl glycogen solution (20mg/ml) to facilitate RNA precipitation. Subsequently, the samples were transferred into the Qiacube instrument and RNA was isolated according to the manufacturers protocol automatically. RNA concentration and integrity were assessed was measured using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA) and RNA quality was measured in a bioanalyzer run using the PicoRNA SmallRNA Chip (Agilent Technologies, Santa Clara, CA, USA).
Label
Cyanine 3-pcp
Label protocol
The expression profile of 2549 human miRNAs was determined using SurePrint G3 8x60k miRNA microarrays (Agilent) according to the manufacturers recommendations
Hybridization protocol
The expression profile of 2549 human miRNAs was determined using SurePrint G3 8x60k miRNA microarrays (Agilent) according to the manufacturers recommendations
Scan protocol
Array´s were scanned with the Agilent Scanner with default settings.
Description
miRNA abundance in serum
Data processing
Raw expression values were extracted using the Feature Extraction Software of Agilent. Raw signal intensities were quantil-normalized and log2 transformed.
