Venous blood samples (5ml) were collected in non-anticoagulant acquisition tubes one day before prostate biopsy for serum harvest (1ml) after coagulation.The blood was centrifuged at 2000 g for 10 min at 4 °C to extract the serum. The serum was stored at -80°C till processed.One milliliter serum was used to isolate EV for each sample. After unfrozen in a 25 °C water bath, the serum was centrifuged at 2000 g for 30 min at room temperature to eliminate residual cell fragments. The serum EV was extracted using Total Exosome Isolation Kit (from serum, Thermo fisher scientific) according to the manufacturer's protocol. After extraction, Exosome Purification and RNA Isolation Kit (Norgen Biotek) was employed to purify EV and extract total RNA in EV consequentially. Agilent-070156 Human_miRNA_V21.0_Microarray platform was used for miRNA-chip assay.
Label
Cy3
Label protocol
Sample tag and reagents are included in the hybrid need Agilent ` s micrornas Complete Labeling and Hyb Kit (p/n 5190-0456), Agilent ` s mirnas Spike - In Kit (p/n 5190-1934) contains two kinds of mirnas Spike - solution In Labeling Spike In solution and Hyb Spike - In solution, used for marking and hybridization reaction In the experimental process quality control
Hybridization protocol
According to Agilent micrornas chip Hybridization standard process and accessory Kit, Gene Expression Hybridization Kit (Cat. # 5188-5242, Agilent technologies, Santa Clara, CA, US), the hybrid furnace in scroll Hybridization Oven (Cat. # G2545A, Agilent technologies, Santa Clara, CA, US) in 65 ℃, 10 RPM, scroll hybrid 17 hours, hybrid cRNA 1.65 mu g / 600 ng, on sample amount and staining in washing cylinder dishes (Cat. # 121, Thermo Shandon, Waltham, MA, US) in the Wash, Wash the slice of the reagents used for Gene Expression for Wash Buffer Kit (Cat. # 5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
Place the chip right in slide holder, place the sample point and face down, and then put it in the scanner. Open the Agilent Scan Control software for 8 x 60 k chips, select AgilentG3_miRNA mode (resolution 3 microns). Set to automatically file access, confirm the scanner status to ready, click Scan Slot m - n, confirm the placement of the chip position. The above steps, when confirmed start scanning chip
Description
serum extracellular vesicle delivered miRNAs of patients with benign prostatic hyperplasia
Data processing
Scanning image Feature Extraction are analyzed with Agilent Feature Extraction (AFE) software version 10.7.1.1 or higher outliers were excluded. Get background subtract and space to trend signal strength for processing. Uneven function markers in the feature extraction function of outliers are excluded.
