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 .
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
50X dNTP stock solution using a 4:1 ratio aminoallyl-dUTP to dTTP***:
**Dissolve 10 mg aminoallyl-dUTP in 170 m L water. Add approx. 6.8 m L 1N NaOH. Final pH is roughly 7.0 using pH paper.
***Altering the ratio of aminoallyl-dUTP to dTTP will affect the incorporation of Cy dye.
1X dNTP final concentration during labeling
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:
Heat to 100C for 2 minutes. Let stand 15 minutes RT.
Apply 38 m L to 40K array. 附件列表→如果您认为本词条还有待完善，请 编辑词条 词条内容仅供参考，如果您需要解决具体问题
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