Revised amino-allyl labeling protocol
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A. RT Reaction
1. To anneal primer, mix 1-2 m g mRNA with 5 ug of anchored oligo-dT [(dT)20 -VN] (Operon, HPLC purified) in a total volume of 18 m L. One reaction for sample mRNA and one for reference mRNA .
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oligo dT
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5 m g of 2.5 m g/ m L
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2 m L
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mRNA /water
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1-2 m g
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16 m L |
2. Heat to 70C for 10 minutes. Cool on ice for 5 minutes.
3. Add 11.6 m L of nucleotide mix to each of Cy3 and Cy5 reactions.
Nucleotide Mix for one reaction
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5X RT buffer
50X dNTP stock solution
DTT
Superscript II RT (Gibco)
RNasin (Gibco, optional)
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0.1M
200U/ m L
40U/ m L
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6.0 m L
0.6
3.0
1.5
0.5
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50X dNTP stock solution using a 4:1 ratio aminoallyl-dUTP to dTTundefined*~I_~K~Hspan~M~K~Hp~M~2~1~Kp~M
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10 m L each 100 mM dATP, dGTP, dCTP (Pharmacia) 8 m L 100 mM aminoallyl-dUTundefined_~ASigma~E_~5A0410~B~K~Hspan~M~K~Hp~M~2~1____________~Kp~M 2 m L 100 mM dTTP
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1X dNTP final concentration during labeling 500 m M each dATP, dCTP, dGTP 400 m M aminoallyl-dUTP 100 m M dTTP
4. Incubate reaction for 1 hour at 42C. Add additional 1 m L reverse transcriptase and continue incubation at 42C for an additional 1 hour.
B. Hydrolysis
1. Degrade RNA by addition of 15 m L of 0.1 N NaOH. Incubate at 70C for 10 minutes 2. Neutralize by addition of 15 m L 0.1 N HCl.
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 from coupling to free amine groups in solution.
3. Add 450 m L water to each reaction.
C. Cleanup
Add 500 m L neutralized, diluted reaction mix to a Microcon-30 filter (Amicon).
Spin at 12g for 7 minutes.
Discard flow through.
Repeat process two more times, refilling original filter with 450 m L water. Concentrate to 10 m L. Samples can be stored at -20C indefinitely.
D. Coupling
Add 0.5 m L 1M sodium bicarbonate, pH 9.0 to 50 mM final. Check 1M stock solution periodically for fluctuations in pH.
Monofunctional NHS-ester Cy3 (PA23001) and Cy5 dye (PA25001, Amersham) is supplied as a dry pellet. Each tube is sufficient to label 10 reactions under normal conditions. Dissolve dry pellet in 20 m L DMSO. Aliquot 2 m L into 10 single use tubes that are then dried in vacuo and store desiccated at 4C. NHS-ester conjugated Cy dye is rapidly hydrolyzed in water, therefore, do not store in DMSO or water. Decreasing the number of aliquots/dye tube may increase your signal.
If you have already made aliquots of dye, simply transfer your cDNA in bicarbonate buffer (10.5 m L) to the aliquot of dye. Alternatively, dissolve Cy dye in 10 ul DMSO and add 1 m L of dye to 10.5 m L of the cDNA reaction. 10% DMSO in the coupling reaction will not affect the chemical reaction. Aliquot unused dye and dry immediately.
Incubate 1 hour at RT in the dark. Mix every 15 minutes.
E. Quenching and Cleanup
Before combining Cy3 and Cy5 samples for hybridization, unreactive NHS-ester Cy dye must be quenched to prevent cross coupling.
Add 4.5 m L 4M hydroxylamine (Sigma).
Let reaction incubate 15 minutes in the dark.
To remove unincorporated/quenched Cy dyes, proceed with Qia-Quick PCR purification kit (QIAGEN). Method described below is as specified by manufacturer.
Combine Cy3 and Cy5 reactions. Add 70 m L water. Add 500 m L Buffer PB. Apply to Qia-quick column and spin at 13K for 30-60 seconds. (optional: reapply flow-though for optimal binding). Decant flow-through. Add 750 m L Buffer PE and spin 30-60 seconds. Decant flow-through. Spin at high speed to dry column. Transfer spin unit to fresh eppendorf tube. Add 30 m L Buffer EB to center of filter and allow to sit 3 minutes at RT. Spin at 13K rpm for 1 minute. Repeat elution step again with another 30 m L of Buffer EB. Pool eluates.
Add 20 m L (20 m g) Cot DNA (Gibco). Add 420 TE and apply to fresh Microcon-30 filter. Spin 12,000g to a volume of 29 m L or less.
For 38 m L array hybridization: 29 m L cDNA probe in TE 1 m L polyA (10 m g; Sigma P9403) 1 m L tRNA (10 m g; Gibco #15401-029) 7 m L 20X SSC 1.2 m L SDS 10%
Heat to 100C for 2 minutes. Let stand 15 minutes RT. Apply 38 m L to 40K array.
