F9, GC-1 and GC-2 cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). The culture dishes for the F9 cells were pre-coated with 0.1 % gelatin.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions.
Label
Cy3
Label protocol
Amplified and labeled cRNA was purified on cRNA Cleanup Module (Agilent Technology) according to the manufacturer’s protocol. Labeled cRNA target was quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE).
Hybridization protocol
After checking labeling efficiency, fragmentation of cRNA was performed by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60oC for 30 min. The fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto assembled Agilent’s Agilent E-array (Teleaulax) (44K). The arrays hybridized at 65oC for 17 hours using Agilent Hybridization oven (Agilent Technology, USA). The hybridized microarrays were washed as the manufacturer’s washing protocol (Agilent Technology, USA).
Scan protocol
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA).
Data processing
All data normalization and further analysis were performed using GeneSpringGX 7.3 (Agilent Technology, USA).
