MEF cells were cultured in DMEM with 10% heat-inactivated FBS (both GIBCO BRL), penicillin (100 U per ml), streptomycin (100 µg/ml), non-essential amino acids (0.1 mM), and L-glutamine (2 mM) in a humidified incubator with 5% CO2 (37°C) iPS-OSH, iPS-J, ESC cultured in DMEM with 15% heat-inactivated FBS (both GIBCO BRL), non-essential amino acids (0.1 mM), L-glutamine (2 mM), 2-Me (0.1 mM), and LIF (103 unit/ml)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by Trizol® Reagent (Invitrogen, USA), and followed by RNeasy Mini Kit (Qiagen, Germany). RNA purified was quantified at OD260nm by using a ND-1000 spectrophotometer (Nanodrop Technology, USA) and qualitated by using a Bioanalyzer 2100 (Agilent Technology, USA) with RNA 6000 nano labchip kit (Agilent Technologies, USA).
Label
Cy3
Label protocol
0.2 g of total RNA was amplified by a Low Input Quick-Amp Labeling kit(Agilent Technologies, USA) and labeled with Cy3 (CyDye, PerkinElmer, USA) during the in vitro transcription process.
Hybridization protocol
0.6 g of Cy3-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60oC for 30 minutes. Correspondingly fragmented labeled cRNA is then pooled and hybridized to Agilent SurePrint G3 Mouse GE 8×60K microarray (Agilent Technologies, USA) at 65°C for 17 h.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 65°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 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
After washing and drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3. Scanned images are analyzed by Feature extraction10.5.1.1 software (Agilent Technologies, USA),
Description
Gene expression of ESC
Data processing
Scanned images are analyzed by Feature extraction10.5.1.1 software (Agilent Technologies, USA), an image analysis and normalization software is used to quantify signal and background intensity for each feature.
