cell type: human embryonic stem cell cell line: H9
Growth protocol
H9 cells were maintained on Matrigel (BD Biosciences) in TeSR-E8 (STEMCELL Technologies). The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Exosomes in the cell culture medium were precipitated by ExoQuick-TC (System Biosciences, EXOTC50A-1). miRNA derived from the exosomes was prepared by using miRNeasy Mini Kit (QIAGEN, 217004) following the manufacturer's recommendations. The miRNA was quantified by a Agilent bioanalyzer, using the RNA 6000 Pico Kit (Agilent, 5067-1513).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 72.4, 100.0, or 51.4 ng of miRNA using miRNA Complete Labeling and Hyb Kit (Agilent, 5190-0456) according to the manufacturer's instructions, followed by MicroBioSpin6 column purification (Bio Rad, 732-62221).
Hybridization protocol
The Cy3-labelled cRNA was dried up with vacuum concentrator, and then was dissolved in hybridization buffer (Agilent, 5190-0456) according to the manufacturer's instructions. The sample was hybridized to SurePrint G3 miRNA Microarray Kit, 8 x 60K (Agilent, G4872A) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute with 37°C GE Wash buffer 2 (Agilent).
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides.
Description
miRNA expression in human embryonic stem cell
Data processing
The scanned images were analyzed with Feature Extraction Software GX14.9.1 (Agilent) using default parameters.
Submission date
Dec 06, 2018
Last update date
Aug 22, 2019
Contact name
Yuzuru Ito
Organization name
National Institute of Advanced Industrial Science and Technology (AIST)
Department
Biotechnology Research Institute for Drug Discovery
Expression profiles of miRNA derived from exosomes for ectoderm-biased substate of human pluripotent stem cells.
Data table header descriptions
ID_REF
VALUE
Signal intensities of <1 were set to 1, each chip was normalized to the 99th percentiles of all measurements from that chips, and the signal was corrected equivalent to 1 ng Baseline transformation of these data was not performed.
