1. Label 15 ml conical tubes according to your timepoints (0, 5, 10, 15, 20 and 30 ).

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1 Harvesting RNA from Yeast for Array Analysis Adapted from Maitreya Dunham, June Hybrid of the DeRisi protocol ( and a standard acid-phenol prep circulating around the Brown/Botstein labs circa RNeasy purification added after experience with student preps in Fall 2006 QCB301. Filtering the cells is the quickest way to collect yeast cells for later RNA processing. Centrifugation is not recommended because it requires more time, during which the yeast cells change their RNA expression. Be sure to have all your tubes labeled and all supplies at hand the more quickly you freeze the cells, the better your gene expression will reflect the moment of harvest. Prepare for sampling. 1. Label 15 ml conical tubes according to your timepoints (0, 5, 10, 15, 20 and 30 ). Assemble filter apparatus. 2. Plug the filter support in a sidearm flask hooked to vacuum 3. Center a 0.45 micron nylon filter on the filter support. (The filter is white, it is separated by blue paper. Use the filter, not paper!) 4. Place the glass funnel on top and carefully clamp it all together. 5. Start the vacuum. 1

2 6. Listen for any whistling noises that may indicate a leak in the seal. If you do get whistling, make sure that the filter is centered and free of wrinkles. Some fraction of the filters have cracks or holes that will interfere with the harvest. Replace the filter with a new one if this seems to be the case. Cell harvest 7. Once everything is ready, remove the culture from the shaker. Turn on the vacuum. 8. Pour 50 ml of culture into the funnel of the filter apparatus. Watch to make sure it is filtering properly and that no cells are making it into the flask. You can refilter if you have this problem, but try to avoid it. 9. Once the culture has completely filtered through, remove the clamp. 10. Then, remove the funnel. 11. Use forceps to lift the edge of the filter, avoiding the cells in the center. 12. Carefully roll up the filter. It is OK to use your gloved fingers to roll up the filter. Speed is more important than neatness! 13. Place the rolled up filter inside the labeled 15 ml tube, loosely cap it, and dunk it into liquid nitrogen. 14. Reassemble the apparatus without a filter and squirt down with water while applying vacuum. Then, reassemble with a new filter to be ready for each sample. 15. After you have collected your 0 timepoint, add 10 ml drug and start your timer. Collect samples at 5, 10, 15, 20, and 30, plunging them immediately into liquid nitrogen. 2

3 Large Scale RNA Prep Your body excretes RNAses (many viruses have RNA genomes, so this RNAse is an evolutionarily good idea but not good for your experiment). To prevent contaminating your experiment with RNase: Before you begin, spray the working area with 0.01% SDS (a much cheaper substitute for RNAse Zap) and wipe it down wear gloves continuously be conscious of everything you touch! When in doubt that you may have touched something contaminated (such as your face, or any surface other than new plasticware), change your gloves, discard your pipet tips, and think clean at all times. Use RNase free reagents and glass-/plastic-ware throughout! Slow down RNAse by keeping RNA on ice at all times (except when the protocol dictates otherwise). RNA lysis 1. Remove samples from liquid nitrogen. 2. Before they thaw, add 4 ml RNA lysis buffer. Vortex, trying to get all the cells off the membrane. 3. Add 4 ml acid phenol (located in fume hood, handle only in the hood.) 4. Vortex. 5. Incubate 15 minutes in 65C waterbath, vortexing every 2 minutes. 6. Incubate on ice 5 min. 7. Spin lysate 5 min 3000 rpm in swinging bucket centrifuge. 8. With a p1000 pipet (not a long plastic pipet, the p1000 gives you better control), transfer the top aqueous layer from the centrifuged samples to the phase lock tube. Discard the tube containing phenol in your phenol waste. 9. Add 4 ml chloroform (located in hood, use glass pipets to handle it polystyrene plastic pipets will melt!). Invert to mix. Do not vortex! 10. Spin 5 min 1500xg (2600 rpm in our Allegra6). The chloroform forms a layer below the phase lock gel, while the aqueous phase containing RNA remains above the phase lock material. 11. Add another 4 ml chloroform to the upper phase of the same tube, invert to mix, and spin again as before. 12. Pour aqueous layer into a new 15 ml Conical tube (not a phase lock tube, a normal empty tube). 3

4 RNA precipitation 13. Add 400 µl 3 M sodium acetate. 14. Mix by vortexing. 15. Add 8 ml (or 2 volumes) 100% ethanol. Mix by inverting. 16. Place at -20 degrees 1 hour (can be overnight). 17. Spin 3000 rpm 10 min. Discard supernatant, saving pellet. 18. Add 4 ml 70% ethanol to wash pellet. 19. Spin 2 min 3000 rpm. 20. Discard wash in waste beaker, and tap tube upside down on paper towel to remove as many drops as possible. 21. Wrap tops of each uncapped tube in parafilm. 22. Poke holes using paperclip. 23. Dry samples in speedvac, using medium heat. Typically this will take about 10 minutes. Dissolving RNA pellet 24. Dissolve pellet in 100 µl water. If yield is very large, you may need to add more, but check with an instructor first. Vortex the tube or flick it to speed dissolution. Examine the tube closely to be sure that the entire pellet dissolves. Once the RNA is dissolved, return it to ice. 25. Your RNA will be too concentrated to accurately quantitate by spectrophotometry. Prepare a 1:10 dilution by mixing 2 ul of your dissolved RNA with 18 ul of nuclease free water. Keep your RNA stock on ice. 26. Measure the concentration of the diluted RNA with the Nanodrop spectrophotometer. 27. Calculate the actual concentration of your RNA (multiply the Nanodrop measurement by your dilution factor). 28. Calculate the amount of RNA from each sample that contains 100 ug. 4

5 RNEasy Cleanup of total RNA for array labeling For each microarray, you will need to purify two RNA samples; one for the labeling reaction, and one for the reference reaction. For time course experiments, a good choice for labeling is a mixed reference. We are providing you a mixed reference sample of RNA from a similar hydrogen peroxide treatment. This RNA has previously been tested for its ability to be labeled and produce good microarray data. You will need to do RNEasy purification of as many reference samples as you have experimental. (Typically, three, if your small group is doing 3 timepoints.) Please note that you will lose 20-40% of the RNA sample during this purification, so you need to begin with more RNA than you will need for the labeling. Also, the maximum binding capacity of the column is 100 ug, so you cannot load more than 100 ug. Exceeding the binding capacity decreases the quality of your RNA. The RNEasy purification dilutes your RNA, so you will re-concentrate it by ethanol precipitation before carrying on with the labeling. Binding RNA to column 1. Prepare a solution of 100 ug total RNA in nuclease free water, so that the total volume is 100 ul. 2. Add 350 ul RLT to the RNA sample, and mix thoroughly by closing the tube and inverting several times. 3. Add 250 ul 100% ethanol to the RNA + RLT sample, and mix by inverting the closed tube. Do not centrifuge. 4. Transfer the RNA-RLT-ethanol mixture (should be approx 700 ul total) to an RNeasy mini column placed in a 2 ml round bottom collection tube. 5. Close the column and centrifuge the assembly for 30 seconds at top speed in an Eppendorf centrifuge. Don t discard the flow through! 6. Transfer the flow through back onto the column. Centrifuge for 30 seconds. This additional step gives a chance for more RNA to bind the column. 7. Transfer the column to a new collection tube. (The RNA is now bound to the column). Discard the used collection tube and flowthrough. 5

6 Washing RNA 8. Add 500 ul buffer RPE to the RNeasy column. 9. Centrifuge for 30 seconds. 10. Add another 500 ul buffer RPE to the RNeasy column. (This is a second wash step.) 11. Centrifuge for 30 seconds. Discard the collection tube and wash buffer. 12. Move the RNeasy column to a clean, empty microcentrifuge tube. 13. Spin the empty RNeasy column 1 minute at top speed. This step is VERY important because it removes traces of the salt and ethanol-containing RPE buffer that can inhibit your reaction. Eluting RNA 14. Move the RNeasy column to a clean, empty microcentrifuge tube. 15. Add 45 ul of nuclease-free water to the RNeasy column. 16. Incubate at room temperature for 1 minute. 17. Centrifuge 1 minute at top speed. 18. Add 45 ul of nuclease-free water to the RNeasy column, leaving the first elution in the bottom tube. (Eluting in two parts increases yield.) 19. Centrifuge 1 minute at top speed. 20. Discard the column, save the combined eluate. Ethanol Precipitation of RNA 21. Add 10 ul 3 M NaOAc to the eluted RNA. Add 200 ul 100% ethanol (NOT 70%!!!). 22. Vortex to mix. (Can store this precipitation at -20 degrees) 23. Centrifuge 10 minutes room temperature at top speed in eppendorf centrifuge. 24. Find the white pellet of RNA (it will be at the bottom of the tube on the side facing the outside of the centrifuge). 25. Remove supernatant with a pipet, being sure not to remove RNA pellet. 26. Add 500 ul 70% ethanol to tube, invert to mix. 27. Centrifuge 5 minutes. 28. Remove supernatant. Tap tubes upside down on clean paper towel to help remove drops, or suck them off carefully with a pipet. 29. Allow pellet to dry COMPLETELY. There should be NO ethanol remaining. You can use the speedvac, or leave tubes open in the hood to dry. Ethanol is VERY BAD for reverse transcription, 6

7 so you must make sure the pellets are dry before resuspending them in water. RNA quantitation and quality check 30. Fully resuspend pellet in 18 ul nuclease free water. Vortex hard and spin down. (If you began with less than 100 ug RNA, adjust resuspension volume appropriately). Keep this RNA on ice. 31. Make a 1:10 dilution by mixing 2 ul of your resuspended RNA with 18 ul water. Check the concentration of this 1:10 diluted RNA using the Nanodrop. (Your RNA stock solution is too concentrated to measure directly.) 32. Check the quality of the RNA on a TAE 1% agarose gel containing ethidium bromide. Combine 4 ul of the diluted RNA sample with 10 ul dye. Run 30 minutes at 75 volts. 33. While the gel is running, calculate the concentration of your RNA by multiplying the concentration of the diluted sample by the dilution factor. 34. Calculate the amount of the RNA stock that contains 50 ug, the amount you will use in the labeling reaction. You will use 50 ug in the labeling reaction. (As little as 30 ug will give acceptable results, if you have less than that you should go back and clean up more RNA.) 35. Check your gel by visualizing on a UV box. If RNA is of good quality, proceed with labeling by reverse transcription. Good quality RNA will have two large, sharp bands of ~1.8 and ~2.5 kb, and a smaller more diffuse band around 200 nt. Bad RNA will be smeary. Yeast total RNA, many samples degraded 7

8 Array analysis of RNA, Labeled via Direct Incorporation During Reverse Transcription Each microarray requires two labeling reactions one experimental and one reference. The input RNA should have been purified by RNeasy. The output of one RNeasy column is required for each labeling reaction. For your experiment, the reference samples provided to you are a mixed reference consisting of RNA collected from several different strains grown under a variety of conditions. The Cy5 dye used for labeling is degraded by ozone, so this work must be done in an area with ongoing ozone treatment, such as the microarray facility or the CIL 003/004 laboratory. Reverse Transcription Reference reaction: set up a single, large scale reaction. Column 1: For 1 rxn ul (contains 50 ug RNA) (14.4 ul volume of RNA used) For reference labeling, multiply column 1 by number of reference rxns Your stock RNA Water to total 14.4 ul per reaction 1 ul 5 ug/ul oligo dt (T 20 VN) 1R. Calculate the amount of RNA necessary for 50 ug per reaction. The maximum volume you can use is 14.4 ul (see 1E) 2R. Calculate the amount of water required so that there are 14.4 ul RNA plus sample per labeling. 3R. Add the calculated amount of anchored oligo dt (T 20 VN). 4R. Put tubes on ice. Experimental reactions: set up one tube for each time point. 1E. Transfer 50 ug of total RNA into a tube. (The maximum volume of RNA you can use is 14.4 ul; if your RNA is not concentrated enough to contain 50 ug in 14.4 ul, as little as 30 ug will give acceptable results. If you have less than 30 ug RNA in 14.4 ul, please discuss it with an instructor.) 2E. Add enough water to total 14.4 ul. 3E. Add 1 µl of 5 µg/µl anchored oligo dt (T 20 VN). 4E. Put tubes on ice. 8

9 Annealing of primer for reverse transcription 5. Incubate in 70 C hot block 10 minutes to denature RNA. During this incubation, prepare the reverse transcriptase master mix (see below). 6. Place samples on ice 5 minutes to allow annealing of anchored oligo dt. Addition of dyes for reverse transcription Reference reaction: 7R. Add Cy3-dUTP to the reference labeling, as calculated below. Column For reference 1: For 1 labeling, multiply rxn column 1 by number of reference rxns 3 ul Cy3-dUTP Experimental reactions: 7E. Add 3 µl Cy5-dUTP to each tube of RNA. Preparation of reverse transcriptase master mix To speed reaction setup and even out pipetting error, it is best to prepare enough reverse transcriptase (RT) master mix for the number of reactions plus one more half reaction (to allow for pipetting error). For example, if you were doing 4 experimental and 4 reference labelings (for a total of 8 reactions), you would set up a master mix containing 6 x 8.5 = 51 ul of 5X superscript buffer, 0.6 x 8.5 = 5.1 ul 50X dntp mix, 3 x 8.5 = 25.5 ul of 0.1 M DTT, and 2 x 8.5 = 17 ul of Superscript II enzyme. You would then use 11.6 ul for each experimental labeling reaction and 4 x 11.6 ul = 46.6 ul for the scaled-up reference labeling (remember that the reference labelings are pooled to make them as consistent as possible). Once you have added the ingredients of your master mix, it is ESSENTIAL that you mix it gently and thoroughly by tapping/flicking the tube and spinning it down in your personal minifuge. Failure to mix the reaction well will mean that your individual tubes won t have the correct proportions of the ingredients. 9

10 Reverse Transcriptase master mix (11.6 µl per reaction) Column 1: For 1 rxn For mastermix, multiply column 1 by number of rxns (reference plus experimental) ul 5X superscript buffer 0.6 ul 50X dntp mix 3 ul 0.1 M DTT 2 ul 200 U/µl reverse transcriptase Note: 50X dntps are prepared from all 4 nucleotides, with lower levels of the labeled nucleotide. In this case, we are using labeled dutp (which is incorporated as dttp), so the unlabeled dttp in the mix is at 40% of the concentration of the other deoxynucleotides. 8. When preparing to set up your master mix, make sure each of the reagents are thawed and well-mixed. Remember to handle all reagents with gloves and keep them RNAse free! For the buffers (5X superscript buffer, the dntps, and the 0.1 M DTT), after they are thawed, vortex each tube well and spin them down. For the reverse transcriptase enzyme, just spin it down NEVER VORTEX ENZYMES, proteins can denature at an air/aqueous interface. 9. After adding all ingredients to a tube, flick tube to thoroughly mix. 10. Spin in your personal quick spin to pool reagent mix in the bottom of the tube. 10

11 Addition of reverse transcriptase master mix Reference reaction: Column 1: For 1 rxn For reference labeling, multiply column 1 by number of reference rxns 11.6 ul Reverse transcriptase master mix 11R. Add correct amount of reverse transcriptase master mix as calculated above. 12R. Flick tube to mix. 13R. Spin in your personal quick spin to pool reagent mix in the bottom of the tube. Experimental reactions: 11E. Add 11.6 µl Reverse transcriptase master mix to each RNA sample, pipetting precisely by transferring 5.8 ul twice using your ul pipet. 12E. Flick tube to mix. 13E. Spin in your personal quick spin to pool reagent mix in the bottom of the tube. 14. Incubate in waterbath 42 C 2 hours After the completion time, stop the reaction by incubating 95 C 5 minutes. (Reactions can be frozen at this point, but in class we will go on to the next step.) 11

12 Reaction Cleanup RNA Degradation (leaving only labeled cdna) Reference reaction: Column 1: For 1 rxn For reference labeling, multiply column 1 by number of reference rxns 15 ul 0.1 N NaOH 1 ul 0.5 M EDTA ph 8 16E. Add the correct amount of NaOH and EDTA as calculated above. Vortex the sample and spin down. (It is very important that the NaOH is well-mixed with the sample so that the hydrolysis of the RNA is complete.) 17E. Incubate 15 min in 65 C waterbath. (RNA is hydrolyzed at high ph and high temperature, leaving only your labeled cdna). Column For reference 1: For 1 labeling, rxn multiply column 1 by number of reference rxns 15 ul 0.1 N HCl Experimental reactions: 16E. Add 15 µl 0.1N NaOH and 1 µl 0.5 M EDTA ph 8. (see below, the reference reaction should be treated at the same time). Vortex the sample and spin down. (It is very important that the NaOH is wellmixed with the sample so that the hydrolysis of the RNA is complete.) 17E. Incubate 15 min in 65 C waterbath. (RNA is hydrolyzed at high ph and high temperature, leaving only the labeled cdna). 18E. Add 15 µl 0.1N HCl to neutralize. 18E. Add the calculated amount of HCl to neutralize the sample. 12

13 Cleanup (purify with Zymo Clean and Concentrator 5 kit) All centrifugation steps are at 13,500 rpm in Eppendorf centrifuge. The small tabletop minicentrifuges are not fast enough for this kit. 19. Carefully label Zymo binding tubes on the small tab, one for each reaction (both reference and labeling). 20. Divide reference labeling reaction into separate tubes corresponding to the number of reactions, 60 ul per tube. This is done because the capacity of the Zymo columns is limited. (You will have an equal number of tubes containing experimental samples and reference samples.) 21. Add 0.5 ml Zymo Binding buffer to each sample, pipet up and down twice to mix. 22. Transfer sample mixture to the column and spin for 30 seconds. If the incorporation has worked well, you will see dye retained on the column. Don t discard the flowthrough yet! 23. Transfer the flowthrough back up to the column and spin for 30 seconds. (This second chance at binding increases yield.) 24. Discard flowthrough from bottom tube by blotting on a paper towel. 25. Add 200 µl wash buffer to column and spin for 30 seconds. 26. Discard flowthrough from bottom tube. 27. Add 200 µl wash buffer and spin for 30 seconds. 28. Discard flowthrough from bottom tube. 29. Replace column onto empty Eppendorf tube. Be careful to space the tubes at least 3 spaces apart so the lids don t interfere with centrifugation. 30. Spin empty tubes 1 minute to remove remaining traces of ethanol. 31. Transfer column to a new Eppendorf tube. 32. Add 22 µl nuclease free water directly to the matrix in the column. 33. Incubate room temperature 1 minute. 34. Spin 1 min to elute. 35. Combine the reference labelings back into a single tube. (Remixing the reactions standardizes your reference among samples as much as possible.) 36. Check yield using Nanodrop. MEASURE THE UNDILUTED SAMPLE! When the Nanodrop program opens, be sure to click on the Microarray button. Then, since you are quantitating a singlestranded cdna, be sure to choose the ssdna value for conversion of A 260 to ug of DNA. You should get in the neighborhood of 1 µg cdna in your 20 ul evolume and observe dye incorporation, typically pmol/ul 13

14 Blocking of microarrays To save time, your arrays have been blocked for you. The blocking procedure for spotted arrays is included at the end of this protocol. Arrays should be blocked the same day that they are used. RNA Hybridization 1. Combine each experimental sample with 20 ul of reference labeling. 2. Add 120 µl water. 3. Add 40 µl 10X Agilent blocking reagent. 4. Add 200 µl Agilent 2X hybridization buffer. Stagger the following steps if you are doing more than 5 arrays, because the timing is important. 5. Incubate 95 C 5 minutes to denature samples. 6. Incubate room temperature 5 minutes. 7. Place a single array backing slide, Agilent side up, in a stainless steel hybridization chamber base. 14

15 8. Pipet the entire volume of probe, avoiding bubbles, onto the center of the gasket area. Don't eject the last µl or two in order to avoid bubbles. 9. Spread the hybridization solution around as you pipet, but do not touch the black rubber gasket. 10. Remove a crosslinked and blocked array from the box. The barcode side is the array side, and so should face down, onto the probe. 11. The barcode must be superimposed over the label on the gasket slide. 12. Carefully lower the array over the gasket slide, keeping the array level and parallel to the gasket slide. 13. Once the array is resting on the gasket slide, place the cover of the hybridization chamber over the array. 14. Leaving the array flat on the table, slide the clamping unit with thumbscrew over the assembly. 15

16 15. Tighten the thumbscrew all the way down, firmly finger tight. 16. Look through the back of the chamber and deliberately and carefully rotate the slide to wet all the gaskets. There should be one big bubble that moves freely. There may be one big bubble and a couple of little ones stuck to the sides. If they are so small they won t affect the array, don t worry too much about them. You will probably do more harm than good trying to remove them. If they seem like they ll block the array from hybridizing, you can try knocking the array vertically against the table to dislodge them. 16

17 17. Put the array in the hybridization oven. Make sure to balance the rotisserie by placing equal numbers of chambers opposite one another (you may need to use an empty chamber). Imagine that the rotisserie is a centrifuge on its side: you balance around the centrifuge, putting samples directly opposite so that the weight around the rotational axis is balanced. If there are few samples, they should go in the middle. 18. Hybridize 65 C overnight at rotation setting Prepare for the washes by placing a bottle containing 500 ml Wash 1 in the 65 C waterbath. Overnight incubation! Array wash and Scanning Washes Wash solutions have been prepared for you. All solutions were filtered through a 0.22 micron filter. Wash 1: 1X SSC, 0.1% SDS Wash 2: 1X SSC Wash 3: 0.1 X SSC 1. Acquire the following: ml beakers, 3 plastic Tupperwaretype containers with lids, 1 vertical slide rack with hook, and 1 horizontal slide rack. 2. Set up a plastic container with enough room temperature Wash 1 to submerge a slide and a 500 ml beaker with a vertical rack (with a bent hook for hanging over edge of beaker) and enough room temperature Wash 1 to cover the racked slides. 17

18 3. Pour the warmed Wash 1 into a 500 ml beaker in the 65 C bath. Place it on top of the submersible stirplate and add a stir bar. 4. Cover the wash buffer with aluminum foil. 5. In the plastic tubs with lids, pour ~500 ml Wash 2 plus a horizontal rack and another with ~500 ml Wash Disassemble each hybridization chamber one at a time. Use the plastic tweezers to gently wedge open the sandwich from the barcode edge while submerged in the chamber of Wash 1. Transfer slide to the rack in RT Wash 1 beaker, with a space between each slide (maximum 6 if you place them back to back). 7. Once all the slides are in the rack, move the rack to the 65 degree Wash1 beaker, hooking the rack on its side. 8. Stir 10 minutes with enough agitation that the solution is visibly turbulent. 9. Transfer slides to the horizontal rack in Wash Shake 10 minutes on the orbital shaker. 11. Transfer rack to Wash Shake 5 minutes on the orbital shaker. 13. Place slide holder on a pad of paper towels in the microplate holders of the tabletop centrifuge. Spin slides dry 2 minutes at room temperature. Buffers RNA Lysis Buffer (100 ml) 2 ml 0.5 M EDTA 5 ml 10% SDS 1 ml 1 M Tris ph ml RNase-free water 50X dntp mix 25 ul 100 mm datp 25 ul 100 mm dttp 25 ul 100 mm dgtp 10 ul 100 mm dctp 15 ul water 18

19 Appropriate stopping points in RNA prep and labeling protocols To help you fit your experiments into available time, there are multiple points at which RNA prep and labeling reactions can be stored: Yeast cells on filters can be stored indefinitely at -80. The ethanol/sodium acetate precipitation step during RNA preparation can be stored at -20. The RNA prep resuspended in water (last step of RNA prep protocol) can be stored at -20 or -80. Following the reverse transcription (labeling) and inactivation step (42 deg 2 hours, 5 min 95 deg), the reaction can be stored at -20. Following the zymo purification step of the labeled cdna, the RNA can be stored before hybridization at -20. If your labeling worked, you will need to prepare the microarrays for labeling by treating them with blocking reagent. Blocking of arrays Arrays should be blocked the same day that they are used. They cannot be stored once blocked, so it is a good idea to wait and see if your labeling worked before you block them. Microarray blocking reagent, 500 ml 500 ml 1.25X SSC (in carboy) 5 ml 10% SDS 2.5 g Roche blocking reagent 1. Put all ingredients in beaker with stirbar. 2. Warm at 65C with stirring (place beaker in 65 degree waterbath with submersible stirbar) until dissolved. 3. Place the arrays in a vertical slide rack. 4. Hang the slide rack on the edge of the beaker and incubate 35 minutes with stirring. 5. Move slides one by one into a new horizontal slide rack in room temperature water. 6. Wash on orbital shaker for 5 minutes. 7. Place slide holder on a pad of paper towels in the microplate holders of the tabletop centrifuge. 8. Spin slides dry at 250 rpm for 2 minutes at room temperature. 19

20 September 2007, adapted from Maitreya Dunham s June 2006 protocol. Modified from various Brown and DeRisi lab protocols. Photographs of Agilent hybridization chambers from Agilent product manuals. 20