Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE buffer 1.For each sample prepare a tube with Extraction Mixture 2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3.Mix and store the sample at -40°C 4.Centrifuge cells at -20°C at 9000 rpm for 10 minutes (Centrifuge RC5B) 5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture 6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7.Freeze the tube immediately in liquid nitrogen 8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 12.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 13.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 14.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at -80°C.
Label
Cy5
Label protocol
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE buffer 1.For each sample prepare a tube with Extraction Mixture 2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3.Mix and store the sample at -40°C 4.Centrifuge cells at -20°C at 9000 rpm for 10 minutes (Centrifuge RC5B) 5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture 6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7.Freeze the tube immediately in liquid nitrogen 8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 12.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 13.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 14.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at -80°C.
Label
Cy3
Label protocol
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
Hybridization protocol
Agilent hybridization protocol for Two-color microarray-based Gene expression analysis. Version 5.5
Scan protocol
Scanned on an ScanArray Express 4000 scanner (Perkin Elmer, Wellesley, MA). Images were quantified using ImaGene Version 7.5 software (BioDiscovery Inc., Marina Del Rey, CA, USA).
Description
Raw data file: slide3-1_2.txt
Data processing
scanned slides were Lowess normalised using Prep (van Hijum, Applied Bioinformatics 2003 2(4):241-244). Interslide scaling based on average intensity of the slide was performed in PostPrep
