1. Add 1 ml Trizol per 5x 106 cells in suspension (1 ml trizol per 10 cm culture disk) and mix by pipetting, transfer to 1.5 ml eppendorf tube. 2. Incubate 5 min at room temperature. 3. Add chloroform (200 µl per ml Trizol), mix for 15 seconds by shaking vigorously. Incubate 2-3 min at room temperature. 4. Centrifuge 20 min at 14000 rpm 4°C. After centrifugation, transfer the water-phase to a new 1.5-ml micro centrifuge tube. 5. add isopropanol to precipitate RNA. Use 0.5 ml per 1 ml trizol. Mix by shaking, leave samples at RT for 10 minutes. Centrifuge at 14000 rpm for 15 min. 6. wash pellet with 75% ethanol. Centrifuge at 14000 rpm for 15 min. Carefully pipet off all ethanol (respin briefly and use a P20 for the last microliters). Airdry pellets briefly before dissolving in MilliQ water. Depending on pellet-size, this may take a while. Continue with RNeasy columns (Qiagen) for clean-up of RNA (maximal 100 µg/column) 7. Adjust each sample to a volume of 300 µl with sterile MilliQ water. Add 160 µl buffer RLT to the samples, and mix (= lysis buffer). 8. Add 240 µl ethanol (100% stored at -20 °C) to the lysate, mix well by pipetting and immediately apply the sample to an RNeasy mini spin column sitting in a 2 ml collection tube (use a second P1000 pipet and work as quick as possible). Next lysate, and so on. 9. Centrifuge at 8000 rpm for 15 seconds in an Eppendorf centrifuge. Discard flowthrough, reuse collection tube. All centrifugation steps at roomtemperature! DNAse treatment: D1. Pipet 350 µl Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm to wash. Discard the flow-through. Reuse the collection tube in step D3. D2. Prepare DNase I incubation mix (80 µl/reaction): add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by gently inverting the tube. Buffer RDD is supplied with the RNase-Free DNase Set. Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex. D3. Pipet the DNase I incubation mix (80 µl) directly onto the RNeasy silica-gel membrane, and place on the benchtop (20-30°C) for 15 min. Note: Make sure to pipet the DNase I incubation mix directly onto the RNeasy silica-gel membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the RNeasy column. D4. Pipet 350 µl Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm. Discard the flow-through and collection tube. 10. Transfer the RNeasy column into a new 2 ml collection tube. Add 500 µl buffer RPE, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse the collection tube in next steps. 11. Pipet 500 µl buffer RPE onto the RNeasy column, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse collection tube. 12. Centrifuge at 10,000 rpm for 2 minutes (to dry the RNeasy membrane). 13. Transfer the RNeasy column into a new 1.5 ml collection tube. Pipet 100 µl milliQ directly onto the RNeasy membrane and incubate for 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. Note: When small yields are expected, the elution volume can be lowered to a minimum of 35 µl milliQ. 14. Pipet the eluate of step 13 again onto the RNeasy membrane and incubate for another 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. After elution, put all RNA-samples on ice. 15. Determine RNA concentration by measuring A260, and RNA intactness by Bioanalyzer. Snap-freeze and store eluate at -80 °C.
Label
Cy5
Label protocol
All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman). mRNA amplification protocol description: * all RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot script. * mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well. * plate is incubated @ 70C for 10 mins, cooled to 48C. * mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and 1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well. * plate is incubated @ 48C for 2 hours and cooled to room temperature. * 106 ul water and subsequently mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH (Promega) is added and mixed in each well. * plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers protocol, to an end volume of 25 ul . * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems). * Plate is incubated @37C for 4 hours. * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C. Labeling protocol description: * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10). All subsequent steps are performed by the robot script : * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines cy3 and cy5 labeled material ). * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells. * 3 ul cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour. * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes. * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers protocol. * RNA concentration and labeling incorporation are measured (SpectraMax 190). * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C. * Samples are stored at -20C until hybridization.
1. Add 1 ml Trizol per 5x 106 cells in suspension (1 ml trizol per 10 cm culture disk) and mix by pipetting, transfer to 1.5 ml eppendorf tube. 2. Incubate 5 min at room temperature. 3. Add chloroform (200 µl per ml Trizol), mix for 15 seconds by shaking vigorously. Incubate 2-3 min at room temperature. 4. Centrifuge 20 min at 14000 rpm 4°C. After centrifugation, transfer the water-phase to a new 1.5-ml micro centrifuge tube. 5. add isopropanol to precipitate RNA. Use 0.5 ml per 1 ml trizol. Mix by shaking, leave samples at RT for 10 minutes. Centrifuge at 14000 rpm for 15 min. 6. wash pellet with 75% ethanol. Centrifuge at 14000 rpm for 15 min. Carefully pipet off all ethanol (respin briefly and use a P20 for the last microliters). Airdry pellets briefly before dissolving in MilliQ water. Depending on pellet-size, this may take a while. Continue with RNeasy columns (Qiagen) for clean-up of RNA (maximal 100 µg/column) 7. Adjust each sample to a volume of 300 µl with sterile MilliQ water. Add 160 µl buffer RLT to the samples, and mix (= lysis buffer). 8. Add 240 µl ethanol (100% stored at -20 °C) to the lysate, mix well by pipetting and immediately apply the sample to an RNeasy mini spin column sitting in a 2 ml collection tube (use a second P1000 pipet and work as quick as possible). Next lysate, and so on. 9. Centrifuge at 8000 rpm for 15 seconds in an Eppendorf centrifuge. Discard flowthrough, reuse collection tube. All centrifugation steps at roomtemperature! DNAse treatment: D1. Pipet 350 µl Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm to wash. Discard the flow-through. Reuse the collection tube in step D3. D2. Prepare DNase I incubation mix (80 µl/reaction): add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by gently inverting the tube. Buffer RDD is supplied with the RNase-Free DNase Set. Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex. D3. Pipet the DNase I incubation mix (80 µl) directly onto the RNeasy silica-gel membrane, and place on the benchtop (20-30°C) for 15 min. Note: Make sure to pipet the DNase I incubation mix directly onto the RNeasy silica-gel membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the RNeasy column. D4. Pipet 350 µl Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm. Discard the flow-through and collection tube. 10. Transfer the RNeasy column into a new 2 ml collection tube. Add 500 µl buffer RPE, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse the collection tube in next steps. 11. Pipet 500 µl buffer RPE onto the RNeasy column, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse collection tube. 12. Centrifuge at 10,000 rpm for 2 minutes (to dry the RNeasy membrane). 13. Transfer the RNeasy column into a new 1.5 ml collection tube. Pipet 100 µl milliQ directly onto the RNeasy membrane and incubate for 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. Note: When small yields are expected, the elution volume can be lowered to a minimum of 35 µl milliQ. 14. Pipet the eluate of step 13 again onto the RNeasy membrane and incubate for another 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. After elution, put all RNA-samples on ice. 15. Determine RNA concentration by measuring A260, and RNA intactness by Bioanalyzer. Snap-freeze and store eluate at -80 °C.
Label
Cy3
Label protocol
All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman). mRNA amplification protocol description: * all RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot script. * mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well. * plate is incubated @ 70C for 10 mins, cooled to 48C. * mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and 1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well. * plate is incubated @ 48C for 2 hours and cooled to room temperature. * 106 ul water and subsequently mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH (Promega) is added and mixed in each well. * plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers protocol, to an end volume of 25 ul . * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems). * Plate is incubated @37C for 4 hours. * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C. Labeling protocol description: * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10). All subsequent steps are performed by the robot script : * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines cy3 and cy5 labeled material ). * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells. * 3 ul cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour. * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes. * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers protocol. * RNA concentration and labeling incorporation are measured (SpectraMax 190). * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C. * Samples are stored at -20C until hybridization.
Hybridization protocol
* 60 ul labeled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA * Hybridizations of spotted oligo-arrays (Codelink glass) or Agilent microarrays are performed on a HS4800Pro Hybstation (Tecan) * Priming: 5xSSC, 0.1%SDS * Probe injection: pre-hyb, 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, Volume 110ul * Hybridization: 45 min at 42C. * Wash 2x: milliQ * Wash: 5xSSC, 0.1%SDS * Probe injection: sample. Volume 110ul (Agilent 4packs: 55ul) * Hybridization: 16 hours at 42C. * Wash 2x: 1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC at 23C * Drying: blow with nitrogen for 3min at 30C
Scan protocol
Scanning of slides using the Agilent G2565BA scanner. Features were extracted using Imagene software from Biodiscovery.
Data processing
Normalization was done using print-tip LOESS as described in (Yang et al. 2002), for all gene probes using no background substraction and a span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve. Signals were corrected for dye bias using intrinsic gene-specific dye biases estimated from the hybset itself, as described in Margaritis, Lijnzaad et al., Mol. Sys. Biol. 2009
