Bauer Core Standard Protocol
Title: Indirect mRNA Labeling and Hybridization
Pages: 5
Revision: 1.0
Date: 8/14/03
Author(s): Claire Reardon
Reviewers: Christian Daly
Contact: claire@cgr.harvard.edu
Comment: This protocol is a modification of a
method provided by Joe DeRisi which was
developed at Rosetta Inpharmatics, Kirkland,
WA. (www.microarrays.org/protocols.html)
1. Purpose
This protocol provides instructions for labeling mRNA for hybridization to microarrays.
The mRNA is labeled with amino allyl dUTP which is then coupled to one of two
Cyanine dyes (Cy3 or Cy5). Cy3- and Cy5-labled probes are combined for competitive
hybridization to the microarray. This method can also be used to label 10–50 µg of total
RNA although this has not yet been optimized at the Bauer Core.
2. Materials
mRNA template(s) and a ready-to-use microarray (already blocked)
Oligo dT12-18 (Invitrogen custom oligo – see section 4.1 for details)
pd N6 (Invitrogen custom oligo – see section 4.1 for details)
Superscript II Reverse Transcriptase (Invitrogen #18064071)
Includes First Strand Buffer (5X) and DTT (0.1 M)
DEPC treated water (Ambion # 9924)
100 mM amino allyl dUTP (Sigma Aldrich # A-0410)
100mM dNTPs (Invitrogen # 10297018)
1N NaOH
0.5 M EDTA (Mallinckrodt # 2580)
1 M HEPES pH 7.5 and pH 7.0 (Calbiochem # 391340)
Microcon YM-30 filters (Millipore # 42410)
Nuclease-free water (Ambion # 9932)
1 M NaHCO3 pH 9 (EM Science # SX0320-1)
Cy 3 mono reactive dye pack (Amersham # PA23001)
Cy 5 mono reactive dye pack (Amersham # PA25001)
DMSO 99.9% (Mallinckrodt # 4948)
QIAquick PCR Purification Kit (Qiagen # 28105)
20X SSC (Invitrogen # 15557036)
7 ug/ul poly(A) (Sigma # P9403) or 7 ug/ul poly d(A)–poly d(T) (Sigma #9764)
0.45 µm Ultrafree-MC filters (Millipore # UFC30HVos)
10% SDS (Invitrogen # 24730-020)
Lifterslips (Erie Scientific -Lifterslip size depends on the size of the array)
Staining Dishes with Slide Racks (Thermo Shandon # 121)
3. Instrumentation
Heat block (e.g. VWR standard)
Hybridization chamber (e.g. Telechem # AHC)
Water bath (e.g. Prescision # 5122 1048)
Centrifuge with plate/slide adapter (e.g. Sorvall Legend RT model)
Microcentrifuge (e.g. IEC Micromax RF)
4. Reagent preparation
4.1. Oligo dT / pd N6 primer
4.1.1. The primer mixture is ordered as custom primers from Invitrogen.
For the Oligo dT order separate dT 12-, 13-, 14-, 15-, 16-, 17- and 18-mer.
Order the pd N6 as NNNNNN.
4.1.2. Resuspend each primer at 20µg/µl.
4.1.3. Mix all of the Oligo dT lengths together at a ratio of 1:1:1:1:1:1:1.
4.1.4. Mix the Oligo dT mix, pd N6 and nuclease-free H2O at a ratio of 1:1:2.
The final concentration Oligo DT and random hexamers is 5µg/µl.
4.2. 50x Amino-allyl dUTP/dNTPs
4.2.1. Resuspend amino allyl dUTP at 100mM in nuclease free H2O.
4.2.2. Mix with stocks of each dNTP for a final ratio of aa-dUTP to dTTP of 2:3.
100mM aa-dUTP
4µl
100 mM dTTP
6µl
100 mM dATP, dGTP, dCTP
10µl each
4.3. Cyanine Dyes
4.3.1. Open one dye pack for each dye (Cyanine 3 and Cyanine 5).
4.3.2. Dissolve the contents of each vial in 20 µl DMSO.
4.3.3. Make 10 aliquots of 2µl in 0.5 ml DNAse-free tubes.
4.3.4. Dry the aliquots using a Speed-Vac under low heat for about 1 hour.
4.3.5. Store the dye aliquots protected from light and desiccated at 4 ˚C until use.
4.4. 3x SSC
Dilute the stock of 20x SSC to 3x.
Add 1.5 ml 20x SSC to a 50ml tube.
Add 48.5ml nuclease free water.
4.5. Wash Buffer 1
Milli-Q H2O
20X SSC
10% SDS
774 ml
24 ml
2 ml
4.6. Wash Buffer 2
Milli-Q H2O
20X SSC
798 ml
2 ml
5. Procedure
5.1 Reverse Transcription (RT) to create amino allyl labeled cDNA.
5.1.1. Mix in a eppendorf tube:
Oligo dT / pd N6
1µl per reaction
mRNA
2µg (0.7 – 4 µg mRNA may be used)
DEPC treated water
to 15.5 µl
15.5µl
5.1.2. Incubate the primer/mRNA mixture at 70˚C for 10 minutes
5.1.3. Chill on ice for 10 minutes.
5.1.4. Make the RT mastermix
5x buffer
6µl
50x aa-dUTP/dNTPs
0.8µl
100mM DTT
3µl
DEPC treated water
3µl
12.8µl
5.1.5. Add 12.8µl RT mastermix to each primer/mRNA mixture.
5.1.6. Add 2.5 µl Superscript II RT enzyme to each reaction.
5.1.7. Incubate at 42˚C for 2 hours (you may incubate up to 3 hours if desired).
5.2. Hydrolysis to remove the RNA template.
5.2.1. To each reaction add and mix well:
1 N NaOH
10 µl
0.5 M EDTA 10 µl
5.2.2. Incubate 15 minutes at 65˚C.
5.2.3. Add 25µl 1 M HEPES pH 7.5 and mix well to neutralize each reaction.
5.2.4. Chill on ice until cleanup.
The cDNA may be stored at –20˚C for up to 2 weeks or at 4˚C over night.
5.3. Cleanup to purify the cDNA and remove the Tris before coupling to the dye.
5.3.1. Label and weigh each microcon-30 filter.
5.3.2. Dilute each cDNA reaction to 500 µl by adding 424ul nuclease-free water.
5.3.3. Add this solution to the microcon-30 column.
5.3.4. Spin at 10,000 xg for 9 minutes and remove the flow-through.
5.3.5. Add 470 µl nuclease free water to the column.
5.3.6. Spin at 10,000 xg for 9 minutes and remove the flow-through.
5.3.7. Repeat the rinse from step 5.3.5.
5.3.8. Spin the column at 10,000 xg until the weight difference is 18 - 20 mg.
(18 - 20µl of solution remains on the filter).
5.4.9. Invert the column into a clean tube and collect at 4,500 xg for 5 min.
The volume collected should be 10 – 13 µl (the filter retains 5 – 7 µl).
5.4. Coupling to the N-hydroxy succinamide ester of the cyanine dye.
5.4.1. Add 1µl of NaHCO3 to the purified probe.
5.4.2. Use the probe resuspend an aliquot of dried cyanine dye (Cy3 or Cy5).
5.4.3. Incubate in the dark at room temperature for 1 hour and 15 minutes.
5.5. Purification and Concentration
5.5.1. Add 70 µl nuclease free H2O to each labeling reaction.
5.5.2. Add 500 µl PB buffer.
5.5.3. Apply the mixture to a Qiaquick PCR purification column.
5.5.4. Spin at 13000 rpm for 45 seconds and remove the flow-through.
5.5.5. Add 700 µl PE buffer to the column.
5.5.6. Spin at 13000 rpm for 45 seconds and remove the flow-through.
5.5.7. Repeat this wash from step 5.5.5.
5.5.8. Spin at 14000 rpm for 1 minute to dry the column.
5.5.9. Transfer to the column to a 0.5 ml tube.
5.5.10. Add 30 µl EB buffer to the filter and let it stand for 1 minute.
5.5.11. Spin at 14000 rpm for 1 minute.
5.5.12. Repeat the elution step with another 30 µl EB buffer.
5.5.13. Label and weigh a microcon 30 filter.
5.5.14. Combine the Cy3 and Cy5 labeled probes and add them to the filter.
5.5.15. Spin the filter for 3 minutes at 10,000 xg
5.5.16. Weigh the microcon and concentrate until the desired volume remains.
5.5.16.1. For 16 pin arrays, spin down to 30 mg (30µl).
5.5.16.2. For 32 pin arrays, spin down to 60mg (60µl).
5.5.16.3. For other sizes of arrays, the probe volume must be optimized.
5.5.17. Invert the filter into a clean tube and spin at 4,500 xg for 5 minutes.
5.6. Hybridization Preparation
5.6.1. To the concentrated probe add:
16 pin arrays
32 pin arrays
20x SSC
3µl
6µl
poly(A) (7 µg / µl)
1.5µl
3µl
1 M HEPES pH 7.0
0.48µl
0.96µl
5.6.2. Wet a Millipore 0.45 µm filter with 10 µl H2O.
5.6.3. Spin at 10,000 xg for 1 minute.
5.6.4. Aspirate the flow-through.
5.6.5. Apply the probe to the filter as a drop.
5.6.6. Spin at 10,000 xg for 1 minute and discard the filter.
5.6.7. Keep the probe at 4˚C until use (up to 12 hours)
5.6.8. Use nitrogen to blow any dust of the microarray
5.6.9. Place the array in a hybridization chamber.
5.6.10. Place a clean Lifterslip over the array with the white strips facing down.
5.7. Hybridization
5.7.1. Add 0.45 µl 10% SDS to the probe.
5.7.2. Heat the probe for 2 minutes at 100˚C.
5.7.3. Allow the probe to cool at room temperatute for 10 minutes
5.7.3.1. Keep the probe protected from light for this time.
5.7.4. Check the volume of the probe.
5.7.4.1. The volume should be 30µl for 16 pin arrays.
5.7.4.2. The volume should be 60µl for 32 pin arrays.
5.7.5.
5.7.6.
5.7.7.
5.7.8.
5.7.9.
5.7.4.3. For other sizes of arrays, the volume must be optimized.
5.7.4.1. Adjust with water if necessary to achieve the desired volume.
Use a pipet to slowly inject the probe under one corner of the lifter slip.
Move the pipet tip along the edge of the lifterslip as you apply the probe.
5.7.6.1. Make sure not to leave any air bubbles under the lifter slip.
Add 100µl 3x SSC to wells at each end of the hybridization chamber.
Seal the hybridization chamber and place in a 62˚C water bath.
5.7.8.1. Keep chamber level during hybridization.
Allow the hybridization to go for 12–16 hours at 62˚C.
5.8. Washing
5.8.1. Fill two staining dishes with Wash Buffer 1 and two with Wash Buffer 2.
5.8.2. Remove the hybridization chamber from the bath, keeping it level.
5.8.3. Wipe the chamber dry and remove the lid.
5.8.4. Remove the slide from the chamber using forceps.
5.8.5. Submerge the slide in Wash Buffer 1 and allow the Lifterslip to fall off.
5.8.6. Place the slide in a slide rack in Wash Buffer 1 and dunk 20 times.
5.8.7. Transfer the rack to a fresh tank of Wash Buffer 1 and dunk 20 times.
5.8.8. Transfer the slide to clean rack and dunk 20 times in Wash Buffer 2.
5.8.9. Transfer the rack to a clean tank of Wash Buffer 2 and dunk 20 times.
5.8.10. Spin the slides dry at 1000 rpm for 2 minutes.
5.8.11. Store the slides in the dark until they are scanned.
5.8.12. Scan within a few hours of washing.