Investigation Title Transcription profiling of human HUVEC cells exposed to 250 uM DETA-NONOate for 4 hours in the presence and absence of 10 uM of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ) Comment[Submitted Name] DETA in HUVEC in absence and presence of ODQ Experimental Design compound_treatment_design transcription profiling by array Experimental Design Term Source REF mo EFO Comment[ArrayExpressReleaseDate] 2005-04-01 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-UMCU-7 Comment[MAGETAB TimeStamp_Version] 2011-06-29 03:45:16 Last Changed Rev: 14857 Experimental Factor Name with or without ODQ Experimental Factor Type dose Experimental Factor Term Source REF Person Last Name Braam de Roos Dijk Kemmeren Holstege Bluysen Koomans Person First Name Branko Remmert Adele Patrick Frank Hans Hein Person Mid Initials Person Email g.b.braam@azu.nl r.deroos@azu.nl a.dijk@azu.nl p.p.c.w.kemmeren@med.uu.nl f.c.p.holstege@med.uu.nl h.a.r.bluysen@azu.nl h.a.koomans@azu.nl Person Phone +31-30-2509111 +31-30-2507329 +31-30-2507329 +31-30-2538959 +31-30-2538186 +31-30-2507329 +31-30-2507329 Person Fax Person Address Person Affiliation Department of Nephrology, UMC Utrecht Department of Nephrology, UMC Utrecht Department of Nephrology, UMC Utrecht Department of Molecular Cancer Research, UMC Utrecht Department of Molecular Cancer Research, UMC Utrecht Department of Nephrology, UMC Utrecht Department of Nephrology, UMC Utrecht Person Roles submitter investigator investigator data_coder;investigator investigator investigator investigator Person Roles Term Source REF Quality Control Type biological_replicate Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2005-04-01 PubMed ID 15699468 Publication DOI 15699468 Publication Author List Braam B, de Roos R, Bluyssen H, Kemmeren P, Holstege FCP, Joles JA, Koomans H Publication Title Nitric oxide-dependent and nitric oxide-independent transcriptional responses to high shear stress in endothelial cells Publication Status journal_article Publication Status Term Source REF Experiment Description HUVECs were exposed to 250 uM DETA-NONOate for 4 hours in the presence and absence of 10 uM of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ). Control cells were exposed to vehicle (methanol 0.1%). Protocol Name P-UMCU-15 P-UMCU-4 P-UMCU-7 P-UMCU-9 P-UMCU-11 Protocol Type grow nucleic_acid_extraction labeling hybridization feature_extraction Protocol Description Title: HUVEC isolation and ODQ exposure. Description: Huvec cells were harvested from freshly obtained umbilical cords. HUVECs were exposed to 250 uM DETA-NONOate for 4 hours in the presence and absence of 10 uM of the guanylate cyclase inhibitor ODQ. Title: Total RNA isolation from mammalian cells. Description: Tissue:
1. 1 ml Trizol reagent for 50-100 mg tissue;

cell culture:
1. 5 ml Trizol reagent for 10 cm2.

2. Incubate 3 minutes at room temperature.
3. Optional (for tissue): pre-heat proteinase K (10 mg/ml) for 10 minutes at 37C. Add 10 ul Proteinase K to the Trizol. Incubate 30 minutes at 55C.
4. Add 0.2 ml chloroform to 1 ml Trizol and shake 15 seconds very well. Incubate 3 minutes at room temperature.
5. Centrifuge 15 minutes 3000 rpm at 4C without brake (15 ml tubes) or 15 minutes 8000 rpm at 4C (1.5 ml Eppendorf tubes). After centrifugation: upper phase = water phase, with RNA; inter phase = DNA and proteins; organic phase = DNA and proteins.
6. Pipette water phase in a new 1.5 ml Eppendorf tube. Use 1 tube per ml Trizol.
7. Add 0.5 ml isopropanol per 1 ml Trizol. Incubate 30 minutes at -20C.
8. Centrifuge 10 minutes at 14000 rpm at 4C.
9. Wash the pellet with 500 ul 70% ethanol.
10. Centrifuge 5 minutes at 14000 rpm at room temperature.
11. Dry and dissolve the pellet in 10 ul RNAse free water. Title: Labeling of RNA. Description: RT REACTION (ratio 7 aa-dUTP : 3 dTTP):

1. Prepare the following RNA/primer mix on ice: 3 ul of mRNA (1 ug/ul, Qiagen oligotex), 6 ul of oligo dT12-18 primer (0.25 ug/ul) and 4 ul of H2O.
2. Incubate at 70C for 10 minutes.
3. After a short spindown chill on ice for 5 minutes.
4. During the 70C incubation of the RNA/primer mix prepare the following labeling mix. (If multiple cDNA synthesis reactions are going to be performed a single labeling mix can be prepared by scaling up the volumes proportionally). On ice add, in the following order: 3 ul of dGAC-mix (1 mM each), 0.9 ul of 1 mM dTTP, 2.1 ul of 1 mM aa-dUTP (Sigma, A-0410), 6 ul of 5X first strand buffer and 3 ul of 0.1 M DTT (total volume = 15 ul).
5. Add the labeling mix to the RNA/primer mix.
6. Incubate at room temperature for 2 minutes.
7. Add 2 ul SuperScript(TM) II Reverse Transcriptase (200 u/ul, Gibco/Life 18064-014). Incubate at 42C for 60 minutes.
NOTES:
1. For specs on the 'ideal target' see Checking Target. 3 ug yeast mRNA gives 300-400 ng target. Other sources of mRNA give different results, e.g. 1 ug of mammalian cell culture mRNA yields 200-300 ng target. In some cases 250 ng of mRNA still resulted in 160 ng cDNA and optimal hybridizations.
2. Prepare the labeling mix just prior to use.
3. Preparation of the RNA/primer and labeling mixes may be scaled up for synthesis of multiple probes. However, the volume of each individual cDNA synthesis reaction should not exceed 30 ul.
4. When a fresh tube of SuperScript(TM) II is opened, change the DTT and 5X first strand buffer as well.
5. Results with total RNA have varied (background on arrays), depending on the source of total RNA, how it was cleaned up, etc. Generally speaking mRNA will give better results and is easier for determining amounts of cDNA and specific yield. 20 ug is a good starting point for total RNA but each source of total RNA needs prior testing on arrays first. Protocol also works well with 5 ug total RNA from biopsy.
6. Unless otherwise specified all water (including water for RNA dilution, measurements, etc) is fresh MQ, autoclaved in a blue-top Nalgene glass bottle.

REMOVAL OF THE RNA TEMPLATE BY HYDROLYSIS:
1. Incubate at 95C for 2 minutes. Chill on ice immediately.
2. Mix together: 10 ul of 1 M NaOH, 10 ul of 0.5 M EDTA, add to the cDNA reaction and incubate at 65C for 15 minutes.
3. After a short spindown, add 25 ul HEPES buffer (1 M, pH 7.5), mixing well.

CLEANUP WITH MICROCON-30 CONCENTRATORS:
To continue with the amino-allyl dye coupling procedure all Tris must be removed from the reaction to prevent the monofunctional NHS-ester Cy-dyes coupling to free amine groups in solution.
1. Fill one Microcon-30 concentrator (Amicon Microcon YM-30, 42410) with 450 ul H2O.
2. Add neutralized reaction.
3. Spin at 10000 g (that is 10000 rpm in Eppendorf 5417C centrifuge) for 8 minutes.
4. Dump flo-thru.
5. Repeat process two times, refilling orginal filter with 500 ul H2O.
6. Elute. Place filter upside down in new tube and spin at 1000 g (3000 rpm) for 3 minutes. Goal is a 30-40 ul eluate. If this is obviously more then spin an extra minute. Adjust protocol next time accordingly. We usually get 25-60 ul eluates. Optional: take 6 ul eluate aside for spectrophotometry (190 to 400 nm range) to determine the amount of cDNA produced. Samples in bicarbonate (next step) may be stored at -20C for at least two weeks prior to coupling.

COUPLING MONOFUNCTIONAL NHS-ESTER CY-DYES:
Note: Cy dyes are not easy to work with. They are sensitive to light, water, perhaps also temperature, pH and freeze-thaw cycles. Avoid ALL possible light. Use dark (amber) Eppendorf tubes. Use aluminium foil. Switch lighting off. Be paranoid.
1. Concentrate samples to 8 ul by using a SpeedVac. Try to keep this step less than 30 min by using a good speedvac. Avoid complete drying of sample.
2. If samples were below 8 ul then add water as required. Add 1 ul of 0.5 M NaBicarbonate buffer, pH 9. Mix well. Proceed with next step only when all samples are ready. Keep on ice when delayed.
3. Resuspend monofunctional NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): Quickly resuspend entire tube in 10 ul DMSO (Merck 8.02912.10). Add 1.25 ul per sample. Mix immediately and quickly. Incubate in the dark at room temperature for 60 minutes. At the moment we aim for 8 reactions at a time so that the whole tube (10 ul) is used. Storage of aliquots is possible by making 1.25 ul aliquots, SpeedVaccing them dry, storing them and using them by adding the 9 ul sample to the aliquot.

QUENCHING:
Before combining Cy3 and Cy5 samples for hybridizations the reactions much be quenched to prevent cross coupling.
1. Add 4.5 ul of 4 M hydroxylamine (Sigma Aldrich 46,780-4).
2. Incubate in the dark at room temperature for 15 minutes.

CLEAN-UP USING CLONTECH CHROMASPIN-30 COLUMNS (DEPC version: K1331-2):
Avoid light contact as much as possible by performing all steps quickly.
1. Upon removing a CHROMA SPIN column from the protective plastic bag, invert it several times to resuspend the gel matrix completely. Before removing the top make sure that all the matrix is at the bottom by vigorously flicking the cloumn downwards to remove matrix that is stuck to the top cap.
2. Holding the CHROMA SPIN column upright, grasp the break-away end between your thumb and index finger and snap off. Place the end of the spin column into one of the 2-ml microcentrifuge (collection) tubes provided, and lift off the top cap. Save the top cap and the white-end cap.
3. Centrifuge at 700 g for 5 min (2700 rpm in Heraeus Megafuge 1.0 Sepatech Centrifuge, 1500 rpm in Eppendorf 5810 centrifuge (prog. 5)). After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration buffer from the column and reestablishes the matrix bed. Make sure that the matrix bed is flat.
4. Remove the spin column and collection tube from the centrifuge rotor, and discard the collection tube and column equilibration buffer.
5. Place the spin column into the second 2-ml microcentrifuge tube. Carefully and slowly apply the sample to the center of the gel bed's flat surface. Drop by drop! Do not allow any sample to flow along the inner wall of the column - this will completely mess up the sample.
6. Centrifuge at 700 g for 5 min.
7. Remove the spin column and collection tube from the rotor and detach them from each other. The purified sample is at the bottom of the collection tube. Immediately store on ice and dark! Purified sample is about 15-30 ul. Bring sample up to 35 ul with water and take 6 ul eluate aside for spectrophotometry (190 to 750 nm range) to determine the amount of cDNA produced and the frequency of dye incorporation. Samples may be stored at -20C for at least two weeks prior to use.

CONTROL OF TARGETS:
The amount of cDNA generated can be checked at two stages: prior to coupling (optional) and after coupling. All targets should have their (specific) activities and yields determined at least prior to use. Because of variations in the amount of background at OD260, 550 and 649 nm it is absolutely essential that proper negative controls are included in the reactions to correct the determinations for this background. Once it has been determined that a particular source of RNA and/or labeling protocol ALWAYS has negligible background values, then these control reactions can be left out. At present we always include a negative control. An ideal control is minus RT enzyme (2x, one for each dye). For comparison of experiments all identical types of measurements should be carried out with the same cuvette/spectophotometer/dilution water combinations. The cDNA measurements below are all done in a 5 ul, 0.5 cm pathlength cuvette on the Shimadzu UV1240mini spectrophotometer. There is a big advantage in determining the full spectrum of absorptions as opposed to single wavelength measurements. For a cDNA only spectrum measure from 190-400 nm. To include the Cys, measure 190-750 nm. Use an identical batch of elution water for blank (or use the negative control itself). Amount of cDNA: OD 260nm x 37 x total volume of probe (ul) = ng of probe. If pathlength of cuvette is 0.5 cm then multiply the result by 2. Subtract the negative control (which means it is best to have equivalent elution volumes)! pmol of dye incorporated: Cy3 OD 550nm x (total volume of probe)/0.15 = pmol of Cy3 dye incorporated; Cy5 OD 649nm x (total volume of probe)/0.25 = pmol of Cy5 dye incorporated. If pathlength of cuvette is 0.5 cm then multiply the result by 2. Subtract the negative control (which means it is best to have equivalent elution volumes)! Frequency of incorporation (# dye labeled nucleotides per 1000 nucleotides): (pmol of dye incorporated x 324.5)/(ng of probe). For full slides (i.e. 3/4 surface coverslip) use at least 200 ng of each probe. 100 ng also works, but more is better, up to 300 ng as far as we know. Use at least 15 pmol incorporated dye. 10 pmol also works but is weaker. Using more than 35 pmol is probably asking for trouble. For smaller coverslips (1/4 total slide surface), use 100 ng target and at least 5 pmol incorporated dye. Frequency of incorporation should be between 20 and 50. 50 is probably rather high. In practice we never go higher than 40. Match the amounts of cDNA for Cy3 and Cy5 on a single slide rather than the activity. Sometimes (batch dependent?), 0.4-0.3 lower pmol amounts of Cy5 give equal intensities on our scanner. Title: Hybridization protocol for gene arrays. Description: PREHYBRIDIZATION:
Make 100 ml prehybridization buffer (for a maximum of 4 slides) containing 5xSSC, 25% formamide (Merck Cat# 1.09684.10), 0.1% SDS and 1% bovine serum albumin (BSA; Sigma Cat# A-9418): 25 ml 20x SSC, 25 ml pure formamide, 49 ml ddH2O, 1 ml 10% SDS and 1 g BSA. After BSA has dissolved the solution becomes clear in about 5 minutes. Filter prehybridization buffer through a 0.22 micron syringe filter. Heat the prehybridization solution to 42C in a 100 ml Coplin Jar. Place slides to be analyzed into the preheated prehybridization buffer. Incubate for 45 minutes at 42C. Wash the slides by dipping 5 times in room temperature ddH2O (staining dish). Dip the slides 5 times in room temperature isopropanol (staining dish). Air dry in fume hood (15 ml tube rack upside down). Slides should be used immediately following prehybridization.

HYBRIDIZATION:
(NOTE: The first steps of the hybridization protocol can be completed during the prehybridization step.) The hybridization volume for a whole slide (25x60mm) is 45 ul. Maximum volume of Cy3 and Cy5 combined target is 25 ul! It could be necessary to concentrate in a speed vac until the volume is below 25 ul. Prepare 2x hybridization buffer containing 50% formamide, 10xSSC and 0.2% SDS: 2.5 ml formamide, 2.5 ml 20xSSC and 0.1 ml 10% SDS. Immediately filter the hybridization buffer through a 0.22 micron filter (before SDS precipitates out of solution). Take 250 ul of the 2x hybridization buffer. Add 5 ul Herring sperm DNA (stock 10 ug/ul, sheared). Final concentration in 2x hybridization buffer is 200 ug/ml. In case of Human Gene Arrays add also 5 ul tRNA (stock 20 ug/ul). Preheat to 42C to overcome SDS precipitation. Combine 25 ul target with 25 ul 2x hybridization buffer (preheated to 42C). Heat at 95C for 5 minutes. Spin at 12K for 2 minutes. DO NOT place on ice after the 2 minute spin. Using standard coverslips: To each array add a total of 45 ul target as 3 to 6 separate dots in the center, along the length of the slide. Put a coverslip (25x60mm) on the array by holding the coverslip in place with your fingertips, toward the bottom of the slide. Gently (!) allow the coverslip to drop onto the array by using a needle. Do not move the coverslip after it is in place. Using LifterSlips (Erie Scientific): Place clean LifterSlip, teflon side down, over the array area. Place the pipette tip along an open edge of the LifterSlip and slowly pipette 45 ul target out of the tip. Capillary action will draw the probe mix under the slip. Place the slide in a Corning Hybridization Chamber (Corning Cat# 2551). Add 20 ul of water to each well in the chamber and clamp the hybridization chamber closed. Gently place the hybridization chamber into a 42C water bath for 16-20 hours.

WASHING THE HYBRIDIZED ARRAYS:
Remove the slide from the hybridization chamber, taking care not to disturb the coverslip. Perform the following washes: (i) Low-stringency wash: Place the slides in a Coplin Jar containing 100 ml 1xSSC and 0.2% SDS (= 100 ml 1xSSC and 2 ml of 10% SDS). Gently remove the coverslip while the slide is in solution. LifterSlips are reusable and should be washed in 70% EtOH prior to use. Incubate for 4 minutes at room temperature. (ii) High-stringency wash: Place the slides in a Coplin Jar containing 100 ml 0.1xSSC and 0.2% SDS (= 10 ml 1xSSC, 90 ml water and 2 ml of 10% SDS). Incubate for 4 minutes at room temperature. Wash the slide finally in 100 ml 0.1xSSC to remove particles of SDS. Incubate for 4 minutes at room temperature. Allow the slides to dry by centrifuge 1 minute 500 rpm (do this immediately after the third wash). Title: Analyses of Microarray images using Imagene 4.0. Description: Protocol Parameters Number of pixels for background buffer;Number of pixels for background area;Number of subgrids per column;Number of subgrids per row;Max size of feature in pixels;Min size of feature in pixels;Number of columns per subgrid;Number of rows per subgrid;Exclude lower percentage of background;Exclude lower percentage of background;Exclude higher percentage of signal;Exclude lower percentage of signal; Protocol Hardware Protocol Software Imagene v4.0 Protocol Contact Protocol Term Source REF SDRF File E-UMCU-7.sdrf.txt Term Source Name mo The MGED Ontology ArrayExpress mo EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version