Plasma from sepsis patients and healthy controls were diluted 1:2 with cold PBS and followed by centrifugation at 2000 g for 30 minutes to remove cellular debris. The supernatant was then diluted 1:5 with PBS and centrifuged at 14,000 g for 30 minutes at 4℃ to remove large membrane vesicles. followed by centrifugation for 2 h at 120,000 g at 4°C in a swinging bucket rotor (Optima XPN- 80, SW 32 Ti rotor, Beckman Coulter). The sediment was washed by PBS again at 120,000 g at 4°C for 2 h.
Extracted molecule
total RNA
Extraction protocol
Plasma EVs were harvested and preserved with Trizol.RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
The labeled cRNA over the procession of inspissation and desiccation and then redissolved with water. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60°C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent),and add the images (.tif) to be extracted to the FE Project,then set FE Project Properties.
