TIG3 diploid fibroblasts were were grown in DMEM media supplemented with 10% (v/v) fetal calf serum. For siRNA experiments cells were transfected with siRNA oligonucleotides specific for EED and harvested for preparation of RNA after 44 hours.
Growth protocol
TIG3 diploid fibroblasts were were grown in DMEM media supplemented with 10% (v/v) fetal calf serum. For siRNA experiments cells were transfected with siRNA oligonucleotides specific for EED and harvested for preparation of RNA after 44 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy mini/midi Kit (RNeasy Mini/Midi Kit, Qiagen). For each siRNA transfection, an RNA pool was obtained by mixing equal quantities of total RNA from each of the six independent RNA extractions.
Label
Biotin
Label protocol
Biotin-labelled cRNA targets were synthesized starting from 5µg of total RNA. Double stranded cDNA synthesis was performed with GIBCO SuperScript Custom cDNA Synthesis Kit, and biotin-labelled antisense RNA was transcribed in vitro using Ambion’s In Vitro Transcription System, including Bio-11-UTP and Bio-11-CTP (NEN Life Sciences, PerkinElmer Inc, Boston, Massachusetts, USA) in the reaction. All steps of the labelling protocol were performed as suggested by Affymetrix (GeneChip® Expression Analysis Technical Manual ). The size and the accuracy of quantitation of targets were checked by agarose gel electrophoresis of 2µg aliquots, prior to and after fragmentation. After fragmentation, targets were diluted in hybridisation buffer at a concentration of 150µg/ml.
Hybridization protocol
Hybridization procedures and parameters: Hybridization mix for target dilution (100 mM MES, 1 M [Na +], 20 mM EDTA, 0.01% Tween 20) was prepared as indicated by Affymetrix, including pre-mixed biotin-labeled control oligo B2 and bioB, bioC, bioD and cre controls (Affymetrix cat# 900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM respectively. Targets were diluted in hybridization buffer at a concentration of 150µg/ml and denatured at 99°C prior to introduction into the GeneChip cartridge. Three copies of the complete GeneChip HG-U133 set (HG-U133A, HG-U133B) were then hybridized with each biotin-labeled target. Hybridizations were performed for 14-16 hours at 45°C in a rotisserie oven. GeneChip cartridges were washed and stained in the Affymetrix fluidics station following the EukGE-WS2 standard protocol (including Antibody Amplification): 1. Wash 10 cycles of 2 mixes/cycle with Wash Buffer A (6X SSPE, 0.01% Tween 20) at 25°C 2. Wash 4 cycles of 15 mixes/cycle with Wash Buffer B (100 mM MES, 0.1 M [Na+], 0.01% Tween 20) at 50°C 3. Stain the probe array for 10 minutes in SAPE solution (10 µg/mL SAPE in 100 mM MES, 1 M [Na +], 0.05% Tween 20, 2 mg/mL BSA) at 25°C 4. Wash 10 cycles of 4 mixes/cycle with Wash Buffer A at 25°C 5. Stain the probe array for 10 minutes in antibody solution (Normal Goat IgG 0.1 mg/mL, 6. Biotinylated antibody 3 µg/mL, 100 mM MES, 1 M [Na +], 0.05% Tween 20, 2 mg/mL BSA) at 25°C 7. Stain the probe array for 10 minutes in SAPE solution at 25°C 8. Final Wash 15 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
Scan protocol
Genechip array scan Images were scanned using an Affymetrix GeneArray Scanner, using default parameters. The resulting images were analysed using Microarray Suite version 5 (MASv5), Affymetrix cat# 690018.
Description
TIG3 diploid lung embryonic fibroblasts were transfected with siRNA oligos specific for the Polycomb protein EED. After 44 hours expression analysis was performed.
