8 protocols
normalization data transformation protocol
Using Quantile normalization, log2 transformed using GeneSpring GX v11.5.1 software package (Agilent Technologies)
normalization data transformation protocol
Quantile normalization using the GeneSpring GX v11.5.1 software package (Agilent Technologies).
array scanning protocol
The hybridized arrays were washed, fixed and scanned with using the Agilent Technologies Scanner G2505C US10450393.(Parameters: Scanning hardware = OTHER: Agilent Technologies Scanner G2505C US10450393, Scanning software = Scanning software)
nucleic acid hybridization to array protocol
1 ug of each labeled cRNA was fragmented by adding 11 ul 10 × Blocking Agent and 2.2 ul of 25 × Fragmentation Buffer, then heated the mixture at 60 C for 30 min, finally 55 ul 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 ul 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 65C in an Agilent Hybridization Oven. (Parameters: Chamber type = OTHER: Agilent’s SureHyb Hybridization Chambers &12-Bay Hybridization Systems - Nimblegen Systems, Quantity of label target used = 1, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 65)
nucleic acid labeling protocol
The samples used the manufacturers Agilent’s Quick Amp Labeling protocol (version 5.7, Agilent Technologies). (Parameters: Amount of nucleic acid labeled = 1, Amplification = RNA polymerases, Mass unit = Micro gram)
growth protocol
The human liver sinusoidal endothelial cells (LSECs) were purchased from ScienCell (San Diego, CA, USA.). The cells were cultured in RIMI 1640 containing 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 ug/ml streptomycin at 37C in 5% CO2. (Parameters: start time = 48, time unit = hours, min temperature = 37, temperature unit = C, media = RIMI 1640 containing 10% (v/v) fetal bovine serum (FBS))
nucleic acid extraction protocol
For mRNA microarray:Total RNA was harvested using TRIzol (Invitrogen).Briefly, 1 ug of total RNA from each sample was amplified and transcribed into fluorescent cRNA with using the manufacturer’s Agilent’s Quick Amp Labeling protocol (version 5.7, Agilent Technologies). For miRNA microarray:Total RNA was harvested using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions. After having passed RNA quantity measurement using the NanoDrop 1000.(Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)
treatment protocol
LSECs were grown in culture medium supplemented with 10% FBS (depleted of endogenous exosomes by overnight centrifugation at 100,000 g) and were treated with or without 1,000 U/ml IFN-a (PBL Interferon Source, Piscataway, NJ, USA) for 48 h. The exosomes from the cell culture supernatants were isolated by differential centrifugation as follow, at 300g for 10 min, 2,000g for 10 min, 10,000g for 30 min, and 100,000g for 70 min, washed once with PBS, and purified by centrifugation at 100,000g for 70 min.