8 protocols
AccessionType
normalization data transformation protocol
miRNAs that intensities>=50 in all samples were chosen for calculating normalization factor. Median Normalization Method was adopted. Normalized Data=(Foreground-Background)/median*NOTE:1. median value of valid probe intensity (background corrected intensity >= 50 in all samples). 2. Each kind of probe has four repeat spots on the microarray, the values in this table are MEDIAN data of the four repeat spots.
normalization data transformation protocol
miRNAs that intensities>=50 in all samples were chosen for calculating normalization factor. Median Normalization Method was adopted. Normalized Data=(Foreground-Background)/median*NOTE:1. median value of valid probe intensity (background corrected intensity >= 50 in all samples). 2. Each kind of probe has four repeat spots on the microarray, the values in this table are MEDIAN data of the four repeat spots.
array scanning protocol
The slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).(Parameters: Scanning hardware = GenePix 4000B [Axon Instruments], Scanning software = GenePix [Axon Instruments])
nucleic acid hybridization to array protocol
After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.16.0) (Exiqon) according to array manual. The total 25 uL mixture from Hy3TM-labeled samples with 25 uL hybridization buffer were first denatured for 2 min at 95C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal.(Parameters: Chamber type = OTHER: 12-Bay Nimblegen Hybridization Systems, Quantity of label target used = 1, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 56)
nucleic acid labeling protocol
The samples were labeled using the miRCURY Hy3/Hy5 Power labeling kit and hybridized on the miRCURY LNA Array (v.16.0).(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))
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.
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)