Collecting cells Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µll phenol/Chloroform -30 µl10% SDS -30 µl3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µlTE 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) 5.Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665) 6.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 7.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
Annealing Mix x µl RNA preparation (=5µg) y µl nuclease-free water 1 µl Random Nonamers 11 µl TOTAL 1. mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down 2. incubate for 5’ 70°C 3. cool at room temperature for 10’ (annealing) 4. spin down the mixture to the bottom of the tube 5. place on ice Reverse transcription Reverse Transcription Mix 4 µl 5x buffer 2 µl 0.1 M DTT 1 µl dNTP-mix 1 µl AA-dUTP 1 µl TMIII (keep only briefly outside 20°C) 20 µl TOTAL 1. Mix by very gently by pipetting up and down (vigorous pipetting will denature the enzyme!) 2. Incubate for 3 hrs at 42°C (in PCR-machine) 3. Cool on ice for immediate purification or place at 20°C for storage Degradation of mRNA 1. Add 2 µl 2.5 M NaOH 2. Mix (vortex) and spin down 3. Incubate for 15 min. at 37°C 4. Add 10 µl 2 M HEPES free acid 5. Mix (vortex) and spin down 6. Ready for purification or store at 20°C Labelling and purification of cDNA with cyanine dyes Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA and Aminoallyl-aRNA" code RPN5661 Labelling of amino allyl-modified cDNA with CyDye 1. Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube 2. Incubate at room temperature, in the dark for 60 to 90 minutes 3. Add 15 µl 4 M Hydroxylamine to each coupling reaction 4. Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 5. Proceed directly to purification of CyDye-labelled cDNA Purification of CyDye labelled cDNA Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA
Collecting cells Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µll phenol/Chloroform -30 µl10% SDS -30 µl3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µlTE 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) 5.Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665) 6.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 7.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
Annealing Mix x µl RNA preparation (=5µg) y µl nuclease-free water 1 µl Random Nonamers 11 µl TOTAL 1. mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down 2. incubate for 5’ 70°C 3. cool at room temperature for 10’ (annealing) 4. spin down the mixture to the bottom of the tube 5. place on ice Reverse transcription Reverse Transcription Mix 4 µl 5x buffer 2 µl 0.1 M DTT 1 µl dNTP-mix 1 µl AA-dUTP 1 µl TMIII (keep only briefly outside 20°C) 20 µl TOTAL 1. Mix by very gently by pipetting up and down (vigorous pipetting will denature the enzyme!) 2. Incubate for 3 hrs at 42°C (in PCR-machine) 3. Cool on ice for immediate purification or place at 20°C for storage Degradation of mRNA 1. Add 2 µl 2.5 M NaOH 2. Mix (vortex) and spin down 3. Incubate for 15 min. at 37°C 4. Add 10 µl 2 M HEPES free acid 5. Mix (vortex) and spin down 6. Ready for purification or store at 20°C Labelling and purification of cDNA with cyanine dyes Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA and Aminoallyl-aRNA" code RPN5661 Labelling of amino allyl-modified cDNA with CyDye 1. Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube 2. Incubate at room temperature, in the dark for 60 to 90 minutes 3. Add 15 µl 4 M Hydroxylamine to each coupling reaction 4. Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 5. Proceed directly to purification of CyDye-labelled cDNA Purification of CyDye labelled cDNA Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA
Hybridization protocol
Two individual differentially , labeled cDNAs were , incubated at 95° C for 3' , cooled down to 68° C and , mixed (final-volume 20 µl). , To these mixed cDNAs , 180 µl of pre-heated (68° , C) Slidehyb#1 hybridization , buffer (Ambion Austin , USA) was added and the , resulting solution was , applied on a pre-heated , slide (68° C). Slides were , then hybridized at 44° C for , 16 hours. Subsequently , slides were washed at , 42°C once in 1 x , SSC/0.2% SDS and twice , in 1 x SSC and dried by , centrifugation (1 x SSC is , 0.15 M NaCl and 0.15 M , Sodium Citrate).
Scan protocol
Using the Scan Array , Express microarray , scanner. 1.Turn on the , scanner and the computer , (in this order) 2.Login: , scanner 3.Double click the , 'ScanArray Express' icon , 4.Switch on lasers 1 and 3 , at least 15 minutes prior to , scanning arrays 5.Click the , 'File' button This is to , prevent you from , accidentally turning off the , laser after the 15 minutes , warming-up phase 6.Insert , the slide with the array-side , up and the label towards , the outside. On Agilent , slides the array side is the , side with the label that has , the word Agilent"" on it , 7.Press 'Scan | Prescan' , 8.Put resolution at 50 m , select both labels used , and set PMT values for , both channels 9.Press , 'Start' 10.Press 'Palette ' , 'Green' as soon as , projection of the image has , started (first dye) select , the red colour as soon as , scanning of the second , layer (second dye) has , started 11.Adjust PMT , values to balance signals , obtained for both , channels. If necessary do , a few low-resolution scans , to obtain a optimal balance , 12.Click the 'Scan' button , again 13.Select frame for , high resolution scan adjust , resolution (to 10 m) and , scan the array for both , dyes 14.Press 'File | Save' , to save the files 'Save all' , saves both dye layers ," Create a new folder for , each array 'Save all' , saves the signals from both , layers in individual files , 15.Switch off the lasers
Description
L_GluExpF1pNZ7021vsGluGlyExpF3pNZ7021
Data processing
the VALUE column is calculated as log2 of ratio (SIGNAL_CH1/SIGNAL_CH2). SIGNAL_CH1 is calculated as F_CH1_MEAN - B_CH1_MEAN and SIGNAL_CH2 is calculated as F_CH2_MEAN - B_CH2_MEAN.
