|Public on Jul 01, 2013
|Liver cells from E15.5 mice
|tissue: CD45- Ter119- liver cells
|RNA was prepared using the RNeasy Mini Purification kit (QIAGEN, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy Mini column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65Â°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37Â°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|Slides were scanned immediately after washing on the Agilent High-Resolution Microarray Scanner using one color scan setting for 4x44k v2 array slides.
|The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
The raw signal intensities of samples were log2-transformed and normalised by the quantile algorithm.
(A total of 16,523 orthologs across the human 4x44K v.2 Agilent oligonucleotide array, and the mouse 4x44K v.2 Agilent oligonucleotide array.)
|May 03, 2013
|Last update date
|Jul 01, 2013
|Yokohama City University
|Graduate School of Medicine
|Department of Regenerative Medicine
|3-9 Fuku-ura, Kanazawa-ku
|Vascularised and functional human liver from an iPSC-derived organ bud transplant