Hepatocytic differentiated cell line MMH-D3 characterised by L. Amicone et al. (Amicone L, Spagnoli FM, Spath G, Giordano S, Tommasini C, Bernardini S, De Luca V, et al. Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes. Embo J 1997;16:495-503).
Growth protocol
Primary cultures were performed in accord to previously described protocol (Amicone L, Spagnoli FM, Spath G, Giordano S, Tommasini C, Bernardini S, De Luca V, et al. Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes. Embo J 1997;16:495-503). Briefly, livers from 14 dpc foetuses and 6 days newborn mice were dissected, mechanically dissociated and plated at high density on collagen-I (Transduction Laboratories, Lexington, UK) coated dishes (Falcon-BD, Franklin Lakes, NJ) in RPMI-1640 (Gibco, Carlsbad, CA), supplemented with 10% Foetal Bovin Serum (FBS) (Gibco), 50ng/ml Epidermal-Growth-Factor, 30ng/ml Insulin-like-Growth-Factor-II (PeproTech Inc, Rocky Hill, NJ), 10µg/ml insulin (Roche, Mannheim, Germany), 2mmol/L L-glutamine, 100u/mL penicillin and 100µg/mL streptomycin (Gibco). The cultures were washed and maintained without transfer for several weeks.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer.
Label
biotin-labeled
Label protocol
One-cycle target labeling assays were performed, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA). Briefly, biotin-labeled target synthesis was performed, starting from 5 µg of total cellular RNA, according to the protocol supplied by the manufacturer (Affymetrix, Santa Clara, CA). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation.
Hybridization protocol
The fragmented cRNA was then hybridized to an identical lot of Affymetrix Mouse430_2 GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE WS2v5 protocol, using antibody-mediated signal amplification.
Scan protocol
GeneChip were finally scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
Description
Synthesis of biotinylated cRNA targets, arrays hybridization (Mouse430_2 GeneChip, Affymetrix, Santa Clara,CA), staining and scanning were performed using Affymetrix standard protocols starting from 5μg of total RNA.
Data processing
Scaling to TGT value=200. The GeneChip-Operating-Software (GCOS) absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples.
