normalization data transformation protocol
Manually flagged bad spots were eliminated and the local background was subtracted, followed by the averaging of replicate features on the array. Log2 intensity ratios (M values) were normalized using Lowess smother.
array scanning protocol
Scanning of slides using the Agilent G2565AA DNA microarray scanner: >Resolution: 10 um >Red laser PMT= 30%, Green laser PMT= 30% >Feature extraction was performed with QuantArray v3.0 software, using the Histogram method for measuring the Mean signal intensity and Mean background intensity.For background calculations were considered the 5-20 percentile of least intense pixels and for signal calculations the 80-95 percentile of most intense pixels. The feature extraction grid was adjusted manually and visually bad spots were flagged using the software flag tool. (Parameters: Scanning hardware = G2565AA DNA microarray scanner [Agilent], Scanning software = QuantArray [PerkinElmer])
PREHYBRIDIZATION: >1. 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. >2. Filter prehybridization buffer through a 0.22 micron syringe filter. >3. Heat the prehybridization solution to 42C in a 100 ml Coplin Jar. >4. Place slides to be analyzed into the pre-heated pre-hybridization buffer. Incubate for 45 minutes at 42C. >5. Wash the slides by dipping 5 times in room temperature ddH2O (staining dish). >6. Dip the slides 5 times in room temperature isopropanol (staining dish). >7. 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 500 ul if using Agilent SureHyb technology or 80 ul if using LifterSlips (Erie Scientific). Maximum volume of Cy3 and Cy5 combined target is half the total hybridization volume. > >1. 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. >2. Immediately filter the hybridization buffer through a 0.22 micron filter (before SDS precipitates out of solution). >3. Take 250 ul of the 2x hybridization buffer and add 5 ul Herring sperm DNA (stock 10 ug/ul, sheared). Final concentration in 2x hybridization buffer is 200 ug/ml. >4. Pre-heat to 42C to overcome SDS precipitation. Combine target with hybridization buffer (preheated to 42C) in a 1:1 proportion. Heat at 95C for 5 minutes. Spin at 12000 rpm for 4 minutes. DO NOT place on ice after the 4 minute spin. > >Using Agilent gasket slides (G2534-60003), Agilent SureHyb hybridization chambers (G2534A) and hybridization oven (G2545A), see Agilent User Manual for instructions: >1. For each array, put a gasket slide onto a SureHyb chamber and add a total of 500 ul target as 1 dot in the center of the chamber. >2. Put a microarray slide on the gasket slide by holding it in place with your fingertips, towards the top edge of the gasket chamber. >3. Gently (!) allow the microarray slide to drop onto the gasket chamber by lowering it with the aid of your other hand. Do not move the microarray slide after it is in place. >4. Place the SureHyb chamber into the hybridization oven at 42C for 16-20 hours. > >Using LifterSlips (Erie Scientific): >1. Place clean LifterSlip, teflon side down, over the array area. >2. Place the pipette tip along an open edge of the LifterSlip and slowly pipette 80 ul target out of the tip. Capillary action will draw the probe mix under the slip. >3. Place the slide in a Corning Hybridization Chamber (Corning Cat# 2551). >4. Add 20 ul of water to each well in the chamber and clamp the hybridization chamber closed. >5. Gently place the hybridization chamber into a 42C water bath for 16-20 hours. > >WASHING OF THE HYBRIDIZED ARRAYS: >1. Remove the slide from the hybridization chamber, taking care not to disturb the coverslip. >2. Separate the slide form the cover while submersed in Low-stringency wash buffer (see below), inside a staining dish (250 ml buffer may be used to disassemble up to 6 hybridizations). Gently remove the coverslip while the slide is in solution. LifterSlips and Agilent gasket slides may be reused up to 4 times and should be washed in 70 % Ethanol prior to use. >3. Perform the following washes: >(i) Low-stringency wash: Place the slides in a Wheaton staining Dish (118mm x 88 mm x 70 mm, Wheaton ref. 900303) containing 500 ml 1xSSC and 0.2% SDS (= 500 ml 1xSSC and 10 ml of 10% SDS). Incubate for 4 minutes at room temperature, with gentle magnetic stirring. (ii) High-stringency wash: Place the slides in a Wheaton staining Dish (118mm x 88 mm x 70 mm, Wheaton ref. 900303) containing 500 ml 0.1xSSC and 0.2% SDS (= 50 ml 1xSSC, 450 ml water and 10 ml of 10% SDS). Incubate for 4 minutes at room temperature, with gentle magnetic stirring. >(iii) Wash the slide finally in 500 ml 0.1xSSC to remove particles of SDS. Incubate for 4 minutes at room temperature, with gentle magnetic stirring. 4. Allow the slides to dry by centrifuging 3 minute 800 rpm (do this immediately after the third wash).
(Parameters: Chamber type = OTHER: Agilent, Quantity of label target used = 300, Mass unit = Nano gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42)
RT REACTION (ratio 7 aa-dUTP : 3 dTTP): >1. Prepare the following RNA/primer mix on ice: 3 ul of mRNA (1ug/ul), 6 ul of oligo dT12-18 primer (0.25 ug/ul, Invitrogen Cat. No. 18418-012), and 4 ul H2O. >2. Incubate at 70 C 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, Invitrogen Cat. No. 18064-014). Incubate at 42 C for 60 minutes. > >NOTES: >1. 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 Milli-Q from Milli-Q Synthesis A10 Model. > >REMOVAL OF THE RNA TEMPLATE BY HYDROLYSIS: >1. Incubate at 95 ?C 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 65 ?C 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: >Note: 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 for 10 minutes. >4. Dump flow-through. >5. Repeat process two times, refilling orginal filter with 450 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 40-80 ul eluates. Optional: take 1 ul eluate for spectrophotometry in NanoDrop (190 to 400 nm range) to determine the amount of cDNA produced. Samples in bicarbonate (next step) may be stored at -20 ?C 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. > >1. Concentrate samples to 16 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 16 ul then add water as required. Add 2 ul of 0.5 M Sodium Bicarbonate 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 18 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 9 ul of 4 M hydroxylamine (Sigma Aldrich 46,780-4). 2. Incubate in the dark at room temperature for 15 minutes. Before proceeding to clean-up, add 8 ul of H2O to raise the sample volume to 35 ul. > >CLEAN-UP USING CLONTECH CHROMASPIN-30 COLUMNS (DEPC version: K1331-2): >Note: 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. After centrifugation, the column matrix will appear semi-dry. This step purges the equilibration solution 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 20-35 ul. Take 1 ul eluate aside for spectrophotometry in NanoDrop (190 to 750 nm range) to determine the amount of cDNA produced and the frequency of dye incorporation. Samples may be stored at -20 ?C 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. 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 are all done in a NanoDrop spectrophotometer. 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. > >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 the spectrophotometer is diferent from 1 cm, then divide the result by pathenght). > >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. For smaller coverslips (1/4 total slide surface), use 100 ng target and at least 5 pmol incorporated dye. Frequency of incorporation is usualy between 30 and 70. Match the amounts of cDNA for Cy3 and Cy5 on a single slide rather than the activity.
(Parameters: Amplification = none, Mass unit = Micro gram)
Strains were grown in synthetic media (SC: 0.67% ammonium sulphate-yeast nitrogen base without amino acids, 2% glucose, supplemented with the appropriate selective amino acids).
Early log-phase cultures (A600=0.4) were divided and half of the culture was exposed to 2mM of CoSO4 for 60 min (WT_Co and yap1_Co).
nucleic acid extraction protocol
1. During harvest centrifugation, accurately determine OD600. For the normalization it is important that all steps are as quantitative as possible (i.e. always yielding identical amounts of total RNA per OD600 of a particular strain under a particular condition). [NOTE: actual numbers may vary from the yields listed here.] >2. Harvest cells by brief (< 4 min) single step centrifugation. Pour off and then aspirate remaining supernatant quickly. Immerse tube in liquid nitrogen immediately. (We use 50 ml tubes, 3 min at 4000 rpm room temperature, never more than 4 tubes in one run, preferably 2 at a time, it is important that nearly all supernatant is removed but without any loss of cells; it takes us 6 minutes from culture to liquid N). >3. Take frozen cells (-80C) and resuspend in Acid Phenol:Chloroform (from Sigma, 5:1, pH 4.7 put at 65C before use). Use 0.5 ml per 25 OD units (volume of culture X OD600; the volumes listed here assume 25 OD units). Immediately add the same volume of TES buffer (TES: 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). Vortex very hard for 20 seconds (at 20 degree angle) to resuspend pellet. >4. Incubate in water bath for 1 hour at 65C. Vortex hard every 10 minutes (10 sec upright, 10 sec at angle). Make sure tubes don't cool off too much. >5. Vortex hard for 20 seconds and transfer to 1.5 ml Eppendorf tubes. Spin for 20 minutes at 14000 rpm at 4C. >6. Take new Eppendorf tube, fill with 0.5 ml Phenol:Chloroform (5:1, pH 4.7). Add water-phase from point 5. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C. >7. Repeat phase separation with Phenol:Chloroform (5:1, pH 4.7) by adding the water phase from point 6 to 0.5 ml Phenol:Chloroform (5:1, pH 4.7) and repeating point 6. >8. Take new Eppendorf tube, fill with 0.5 ml Chloroform:Isoamyl-alcohol (25:1). Add water-phase from point 7. Vortex hard for 20 seconds. Spin down for 10 minutes at 14000 rpm at 4C. >9. Take new Eppendorf tube, fill with 50 ul Sodium Acetate 3M, pH 5.2. Add water-phase from point 8. Fill tube with Ethanol (98 %, -20C). Incubate at -20C for longer than 30 minutes (usually overnight). >10. Spin tubes down for 5 minutes at room temperature, 14000 rpm. Remove fluid with pipette. Be careful not to touch RNA pellet. >11. Wash pellet with 500 ul Ethanol (80 %, -20C). Spin down briefly. >12. Remove all traces of Ethanol, air dry for 1 minute. >13. Dissolve RNA pellet in sterile RNAse free water (MilliQ), to a concentration of approximately 10 ug/ul. (Reference yields: 1 OD600 mid-log is equivalent to 40 ug RNA, heat shocked mid-log is lower; 1 OD600 Stationary Phase is equivalent to 10 ug RNA) (Parameters: Extracted product = total_RNA, Amplification = none)
(Parameters: Extracted product = total_RNA, Amplification = none)