Dahlquist:DNA Microarray Protocol
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=== Day 5 ===
=== Day 5 ===
To be continued...
To be continued...
Revision as of 20:46, 9 June 2010
This protocol describes all the steps necessary to perform aRNA synthesis, labeling, hybridization, and scanning of DNA microarrays for Saccharomyces cerevisiae, beginning with total RNA.
NOTE: This protocol is still under construction!
NOTE: RNase-free supplies should be used whenever possible.
- SealRite 1.5 mL Natural Microcentrifuge Tubes (free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1615-5500)
- TipOne 101-1000 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1126-7810)
- TipOne 1-200 μL Graduated Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1120-8810)
- TipOne 0.1-10 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1121-3810)
- 50 ml conical screw cap tubes, copolymer, bulk, sterile (USA Scientific Catalog #1500-1211)
- Powder-free latex exam gloves (USA Scientific Catalog #4900-2200, 4900-3300, 4900-4400)
- multiwipes, small and large (Denville Scientific Catalog #C5709-1A, C5709-3A)
- a.k.a. Kim wipes
- Denville Scientific sells a generic version that is less expensive than the name brand
- aluminum foil (from grocery store or other such supplier)
- compressed air duster
- VWR sells something called the "Whoosh Duster" (VWR Catalog #16650-027), but I've found that one purchased from an electronics store (less expensive) works fine (Kam D. Dahlquist 20:46, 15 August 2008 (EDT))
- trUView™ Disposable Cuvettes (BioRad Catalog #1702510)
- UV-transparant microcuvettes hold 50 μL total volume
- can be cleaned and re-used indefinitely when you don't need to recover the sample from them
- RNaseZap (Denville Scientific Catalog #D1180)
- Actually an Applied Biosystems/Ambion product Catalog #AM9780, but it is less expensive if you buy it from Denville
NOTE: RNAse-free reagents should be used whenever possible.
- Amino Allyl MessageAmp™ II aRNA Amplification Kit (Applied Biosystems Catalog #AM1753)
- CyDye Post-Labeling Reactive Dye Pack (GE Healthcare Life Sciences Catalog #RPN5661)
- TE Buffer (USB Catalog #75893)
- Nuclease-free water (non-DEPC treated)
- Nuclease-free water is provided in the Amino Allyl MessageAmp™ II aRNA Amplification Kit and is sufficient for the μL quantities called for throughout the protocol; additional water can be purchased from Applied Biosystems/Ambion, but it is expensive.
- DEPC is an oxidizer and should be fully removed from water used with DNA microarrays because the Cy5 dye is prone to degradation by oxidation.
- For larger quantities of nuclease-free water (non-DEPC treated) that are needed to dilute 20X SSC for washes, for example, I use Water, ASTM Type II, non-sterile, Reagent Grade, ACS (VWR Catalog #RC91505) that can be purchased in 10 L or 20 L cubes. This water was recommended by Genisphere in the DyeSaver 2 protocol as being validated for use with microarrays and not containing components that will oxidize Cy5; Kam D. Dahlquist 20:24, 15 August 2008 (EDT))
- RNA Fragmentation Reagents (Applied Biosystems Catalog #AM8740)
- Product information
- 200 reactions per kit
- DIG Easy Hyb (Roche Applied Science Catalog #11796895001)
- Fish sperm DNA Solution 10 mg/mL
- Have used Ultra Pure Salmon Sperm DNA Solution from Invitrogen (Catalog #15632-011)
- When that runs out will use DNA, MB-grade from fish sperm from Roche Applied Science (Catalog #11467140001) which is significantly less expensive
- Oligo dA, 10-20mer, 1μg/mL (Invitrogen Catalog #POLYA.GF)
- 100% ethanol (Sigma Catalog #E702-3)
- for adding to wash buffers in Amino Allyl MessageAmp™ II aRNA Amplification Kit
- 20X SSC (USB Catalog #19629)
- for making post-hybridization wash buffers
- 20% SDS (USB Catalog #75832)
- for making post-hybridization wash buffers
- Hybri-slips (Sigma Catalog #H0784)
- pack of 100
- inexpensive, disposable, easy-to-use, made out of plastic
- L × W × thickness 60 mm × 24 mm × 0.25 mm
- Yeast 6.4K Array (Y6.4K) (University Health Network Microarray Centre, Toronto)
- Slide Coating Solution
- "Home-brew" version of Genisphere DyeSaver 2
- 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene
- 200 mL needed to fill Coplin jar
- Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.
- Can be re-used
- Dedicated set of RNase-free pipetmen
- Water bath set to 50°C
- Water bath set to 37°C
- Heat block set to 70°C
- Corning hybridization chambers
- racks (for 1.5 mL microcentrifuge tubes)
- racks (for 50 mL conical tubes)
- ice bucket and ice
- slide box
- glass Coplin jar (to hold Slide Coating Solution)
- UV/vis spectrophotometer
- Use RNase-free reagents and supplies and maintain good RNase-free technique throughout.
- At the beginning of each day's work, clean the bench top and pipetman shafts with RNaseZap.
- Keep all samples on ice unless specifically noted otherwise.
- Once the Cy3 and Cy5 dye packages have been opened, all procedures must be carried out in the dark.
- Generally, we perform up to 10 reactions at one time.
- Make sure to perform multiple reactions for the reference sample so that there is enough aRNA for each of the chips.
- If using kit for the first time, add the appropriate amount of 100% ethanol to the Wash Buffer.
- Dry 1 μg of yeast total RNA to less than 10 μL in a SpeedVac.
- 1 μg is the largest amount recommended for the kit.
- RNA can be dried ahead of time and stored at -80°C.
- Reverse Transcription to Synthesize First Strand of cDNA: follow steps C1-C4 in protocol.
- Can use heat block for 70°C incubation in step C2.
- Use 42°C water bath for step C4.
- Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.
- Second Strand cDNA synthesis: perform steps D1-D2.
- Use 16°C water bath in the cold room for step D2.
- Potential stopping point for Day 1. Freeze samples at -20°C. Protocol says it is better to go on to cDNA purification before freezing, though.
- cDNA Purification: Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 50°C in a water bath (the protocol says to heat the entire bottle, but this is unwieldy).
- Perform steps E1-E4.
- Stopping point for Day 1. Freeze samples at -20°C.
- Perform steps F1-F3 (In Vitro Transcription to Synthesize Amino Allyl-Modified aRNA.)
- Perform step F2 in 37°C water bath for six hours. The protocol says to incubate for 4-14 hours. However, it is important to incubate the same amount of time for each experiment to reduce variability.
- Potential stopping point for Day 2. Freeze samples at -80°C.
- aRNA Purification: Aliquot nuclease-free water needed for elution step (G5) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 50°C in a water bath.
- Perform steps G1-G5.
- Do not vortex and do not centrifuge in steps G1 and G2 and proceed as quickly as possible to step G3, otherwise the aRNA will start precipitating and you will lose it.
- Stopping point for Day 2. Freeze samples at -80°C.
Assess aRNA yield and quality through UV absorbance and agarose gel electrophoresis.
- Make a 1:50 dilution of each aRNA sample by diluting 2 μL of sample into 98 μL of TE Buffer.
- Read absorbance at 260 and 280 nm.
- Calculate the concentration of aRNA using the formula
A260 X dilution factor (50) X 40 μg/mL = concentration (μg/mL) (for 1 cm path length)
- The 260/280 ratio should be between 1.7 and 2.1.
- Calculate and aliquot the volume of aRNA required for 1 μg of aRNA into a fresh 1.5 mL microcentrifuge tube for each sample.
- This could be a stopping point for Day 3, storing the samples at -80°C, but ideally, go on to the next step:
Agarose gel electrophoresis using Reliant RNA Gel System by Lonza
- Bring volume of aRNA to 5 μL using nuclease-free water.
- Add 5 μL of 2X formaldehyde sample buffer (loading dye) (Lonza) to each sample.
- Remove aliquotted sample of 0.2-10 kb RNA markers (USB) from -80°C freezer (dye already added) and include in subsequent steps.
- Incubate 65°C for 15 minutes.
- Incubate on ice for 1 minutes.
- In the meantime, place the pre-cast Reliant gel on the loading tray of the large horizontal gel box.
- Make sure that it is not crooked.
- Fill box to cover gel with 1X AccuGENE MOPS Running Buffer (Lonza).
- Dilute 100 mL 10X AccuGENE MOPS Running Buffer with 900 mL MilliQ water to make 1 L.
- Can be saved and re-used several times (stored at room temperature) for this type of gel.
- Run at 60-65 volts constant voltage for 2 hours.
- Remove gel from casting tray and place in small tupperware container.
- Submerge gel, just to cover, with MilliQ water.
- Add 10 μL of 10,000X GelStar Nucleic Acid Gel Stain (Lonza).
- Gently agitate for 20-30 minutes.
- Detect the RNA using UV transillumination with Kodak Gel Logic 100 station.
- Amplified aRNA should appear as a smear from 250 to 5000 nt. The average size of amino allyl aRNA should be approximately 1400 nt.
- Mix together aRNA samples if multiple reactions were performed for the same total RNA sample and each passes quality control. Re-read absorbance values and re-calculate concentration.
- Calculate the volume required for 20 μg of aRNA and aliquot into a fresh 1.5 mL microcentrifuge for each sample (if you need multiple reference samples, make sure to aliquot enough to hybe all of your chips). Store all samples at -80°C. Stopping point for day 3.
Indirect Labeling by Dye-Coupling
NOTE: this protocol uses the Applied Biosystems (formerly Ambion) Amino Ally MessageAmp II aRNA Amplification kit and GE (formerly Amersham) CyDye single reaction dye pack of the activated Cy3 and Cy5 dyes. The following steps are performed in the dark and are taken from the Ambion protocol.
- Place 20 μg of amino allyl aRNA in a nuclease-free microfuge tube, and vacuum dry to completion on the low setting (takes 30-60 minutes, depending on volume). After 30 minutes, check every 5-10 minutes and remove sample as soon as it is dry; do not overdry.
- To tube containing dried aRNA, add 9 μL Coupling Buffer and mix well/flash spin.
- Resuspend each CyDye pack in 11 μL of DMSO [and keep in the dark at room temperature for up to 1 hour until ready to use it.] You will need one Cy3 and one Cy5 pack per microarray.
- To tube containing the aRNA:Coupling Buffer mixture, add 11 µL of dye to each tube, mix thoroughly and flash spin.
- NOTE: Make sure you know which dye to add to which tube!
- Incubate at room temperature in dark for 30 minutes (Wrap the rack with foil and place the rack in a drawer).
- To quench the reaction, add 4.5 μL of 4 M hydroxylamine and mix well. Incubate at room temperature in the dark for 15 minutes.
- Add 5.5 μL of nuclease-free water to bring each sample to 30 μL.
Removing Uncoupled Dye Material
- Before beginning the procedure, pipet enough nuclease-free water into a fresh tube so that you have 20 μL X number of tubes, plus a little extra. Pre-heat to 50-60°C. The water should heat for at least 10 minutes.
- Add 150 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.
- Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. Do NOT vortex and do NOT centrifuge.
- Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. Spin 1 minute at 10,000 X g. Discard flow-through.
- Apply 500 μL Wash Buffer to each filter cartridge. Spin 1 minute at 10,000 X g. Discard flow-through.
- Spin again at 10,000 X g to remove trace amounts of Wash Buffer.
- Transfer filter cartridge into a fresh collection tube, being careful not to contaminate the new tube with any of the flow through from the previous step.
- To the center of the filter, add 10 μL of the nuclease free water that you pre-heated to 50-60°C in step 1.
- Let sit at room temperature for 2 minutes, then spin for 1.5 minutes at 10,000 X g.
- Repeat the elution with another 10 μL of nuclease free water pre-heated to 50-60°C.
- Let sit at room temperature for 2 minutes, then spin for 1.5 minutes at 10,000 X g.
Determine the concentration of labeled aRNA
- Dilute the labeled aRNA 1:50 by placing 1 μL of labeled aRNA into 49 μL of TE Buffer in a fresh tube.
- Read A260 and A550 for Cy3 or A650 for Cy5. If A260 is below 0.1, adjust dilution and try again.
- A calculator for RNA concentration and labeling efficiency can be found at: http://www.ambion.com/techlib/misc/aama_dye_calc.html
- If you had multiple tubes of reference sample, and they all look OK on the spec, mix them together and re-read them as a single mixed sample.
- Calculate the volume required for 5 μg of Cy3 and Cy5 labeled aRNA. Mix those volumes of the appropriate Cy3 and Cy5 labeled aRNA together.
- NOTE: Be sure to mix the right samples together!
Preparation for hybridization
- Vacuum dry the labeled aRNA that will be used for hybridization in a speedvac until the volume reaches 1-10 μL. Do not overdry.
(This will take ~60-90 minutes.)
- Bring the volume of the dried labeled aRNA to 9 μL with nuclease-free water.
- Add 1 μL of Ambion 10X Fragmentation Buffer to the labeled aRNA.
- Incubate at 70°C for 15 minutes.
- Add 1 μL of the Stop Solution and place on ice.
- Speedvac the fragmented aRNA until the volume is < 5 μL. This will take about 5 minutes. Do NOT overdry.
- Make the hybridization master mix as follows:
X1 X6 DIG Easy Hyb solution 100 μL 600 μL 1 μg/μL oligo dA 0.38 μL 2.28 μL 10 mg/mL sonicated salmon sperm DNA 5 μL 30 μL
- Mix and flash spin. Incubate at 65°C for 2 minutes. Let cool until room temperature.
- Add 85 μL of the hybridization master mix to the paired, dried sample. Incubate at 65°C for 2 minutes. Flash spin and let cool until room temperature.
- Place the cover slip with the smooth side up on a support (a rack for 15 mL test tubes works great). Have the microarray slide ready with the hybridization chamber. Pipet the hybridization solution in a line on the cover slip, avoiding the introduction of air bubbles. Holding the microarray slide only by the edges (do not touch the front or back of the slide), pick up the cover slip with the DNA side of the slide down (the label side is the DNA side) and quickly invert the slide and place in the chamber with the DNA side and cover slip up.
- Pipet 11 μL of nuclease-free water into the wells in the hybridization chamber on either side of the slide. Place the cover on the chamber and lock the clips in place.
- Working quickly and keeping the hybridization chamber horizontal, place the chamber at the bottom of the 37°C water bath. Incubate for 15-16 hours.
Post-hybridization washing and scanning
- Remove the hybridization chamber from the water bath and dry the outside with a large kim wipe. Pull the clamps off and wipe again before opening the chamber. Lift the slide from the barcode end using forceps. Remove the coverslip by quickly, but gently dipping the slide in a 50 mL conical tube filled with room temperature 1X SSC. Let the coverslip slide off gently; hold the slide at the barcode end with forceps. Once the cover slip comes off, transfer the slide to another conical tube filled with 50°C 1X SSC/0.1% SDS. Incubate for 15 minutes in the 50°C waterbath.
- Transfer slide quickly to a second tube filled with 50°C 1X SSC/0.1% SDS. Do not let the slide dry out while transferring. Incubate for 15 minutes in the 50°C waterbath.
- Transfer slide quickly to a third tube filled with 50°C 1X SSC/0.1% SDS. Do not let the slide dry out while transferring. Incubate for 15 minutes in the 50°C waterbath.
- After the three washes are complete, rinse the slide in a tube filled with room temperature 1X SSC by plunging up and down 4-6 times.
- Rinse the slide in a tube filled with room temperature 0.1X SSC by plunging up and down 4-6 times. When you pull the slide out the last time, pull it out very slowly so that water droplets do not form on the surface of the slide. Then carefully blot the edges of the slide dry on a folded kim wipe, being careful not to touch the front or back surface of the slide. Use the compressed air duster to blow off any water droplets that remain. You are not blowing the water dry, but chasing it to the edge of the slide so that it can be blotted by the kim wipe.
- Carry the dry slide in a slide box covered in foil to the fume hood in the Keck Lab. Dip the slide into the coplin jar filled with 2% PEG (2000) in 1:1 acetone:toluene. Be careful not to dip the label into the solvent. Work quickly. Allow the slide to dry for about 1 minute before putting it back in the box and bringing it back into the Dahlquist lab for scanning.
To be continued...
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