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Malaysia University |
REVERSE TRANSCRIPTION AND AMINO-ALLYL COUPLING OF RNA FOR MICROARRAY ANALYSIS OF GENE EXPRESSION
Reverse Transcription and Amino-allyl Coupling of RNA for Microarray Analysis of Gene Expression
Overview
This protocol describes the preparation of fluorescent cDNA from cellular RNA. The fluorescent cDNA is used as a probe to detect gene expression on microarrays. DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray.
Procedure
A. Reverse Transcription Reaction
The first step is to reverse transcribe the RNA into cDNA in the presence of the amino-allyl nucleotide. The incorporated amino groups allow the cDNA to couple to the fluorescent dyes, Cy3 or Cy5. Stop the reaction by base hydrolysis of the RNA template. After neutralization and clean up, the cDNA is allowed to conjugate with the reactive Cy dyes. Once that reaction is quenched and cleaned up, the probe is ready.
1. Anneal the primer by mixing the following reagents in a microcentrifuge tube:
5 μg of anchored oligo-dT
1 to 2 μg of sample mRNA
Adjust total volume to 18 μl with ddH2O
One reaction for the sample mRNA, and one for the reference mRNA.
2. Heat the reaction to 70°C for 10 min.
3. Cool on ice for 5 min.
B. cDNA Synthesis
1. Add 11.6 μl of Nucleotide Mix to each of Cy3 and Cy5 reactions.
5. Incubate the reaction for 1 hr at 42°C.
6. Add an additional 1 μl of Reverse Transcriptase and continue the incubation at 42°C for an additional hr.
C. Hydrolysis
1. Degrade the RNA by adding 15 μl of 0.1 M NaOH.
2. Incubate at 70°C for 10 min.
3. Neutralize the reaction by adding 15 μl of 0.1 M HCl.
4. Add 450 μl of ddH2O to each reaction.
5. 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.
D. Clean-up
1. Add 500 μl of the neutralized diluted reaction mix to a Microcon-30™ filter.
2. Centrifuge at 12,000 X g for 7 min.
3. Discard the flow-through.
4. Repeat the process twice more, refilling the original filter with 450 μl of ddH2O.
5. Concentrate the sample to 10 μl (see Hint #1).
E. Coupling
1. Add 0.5 μl of 1 M Sodium Bicarbonate to a final concentration of 50 mM.
2. Transfer 10.5 μl of the cDNA prepared in Section E, Step #1 to the aliquot of dye (see Hint #2 and Hint #3).
3. Incubate the reaction for 1 hr at room temperature in the dark. Mix every 15 min.
F. Quenching and Clean-up
Before combining Cy3 and Cy5 samples for hybridization, non-reactive NHS-ester Cy dye must be quenched to prevent cross coupling
1. Add 4.5 μl of 4 M Hydroxylamine.
2. Incubate the reaction for 15 min in the dark.
3. To remove unincorporated/quenched Cy dyes, proceed with the Qia-Quick PCR purification kit (QIAGEN). The method described below is as specified by manufacturer.
4. Combine the Cy3 and Cy5 reactions.
5. Add 70 μl of ddH2O.
6. Add 500 μl of Buffer PB [supplied by QiaQuick PCR purification kit (QIAGEN)].
7. Apply to the Qia-quick column and spin at 13,000 X g for 30 to 60 seconds (see Hint #4).
8. Decant the flow-through.
9. Add 750 μl of Buffer PE [supplied by QiaQuick PCR purification kit (QIAGEN)].
10. Centrifuge the column at 13,000 X g for 30 to 60 seconds.
11. Centrifuge again at high speed to dry the column.
12. Transfer the spin unit to a fresh microcentrifuge tube.
13. Elute by adding 30 μl of Buffer EB [supplied by QiaQuick PCR purification kit (QIAGEN)] to the center of the filter and allow it to sit for 3 min at RT.
14. Centrifuge the column at 13,000 X g for 1 min.
15. Repeat the elution step again with another 30 μl of Buffer EB.
16. Pool the eluates.
17. Add 20 μl (20 μg) of Cot DNA (Gibco).
17. Add 420 μl of TE and apply to a fresh Microcon-30™ filter.
18. Follow the manufacturer's instructions and concentrate to a volume of 29 μl or less.
19. For a 40K array, use 38 μl of hybridization mix as follows:
29 μl of cDNA probe
1 μl of poly A
1μl of tRNA
7 μl of 20X SSC
1.2 μl of 10% SDS
20. Heat to 100°C for 2 min.
21. Let stand for 15 min at room temperature.
22. Apply the 38 μl reaction to the 40K array.
Solutions
RNasin
40 Units/μl RNasin (Gibco, optional)
tRNA
10 mg/ml (Gibco #15401-029)
Nucleotide Mix
0.6 μl of 50X dNTP stock solution
6.0 μl of 5X RT Buffer
3.0 μl of 0.1 M DTT
Superscript II RT (Gibco)
200 Units/μl Superscript II Reverse Transcriptase (Gibco)
(for one reaction)
1.5 μl of Superscript II RT (Gibco)
0.5 μl of RNasin
poly A+ RNA
10 mg/ml (Sigma P9403)
Anchored oligo-dT
2.5 μg/μl (dT)20-VN
(Operon, HPLC purified)
4 M Hydroxylamine (Sigma).
1 M Sodium Bicarbonate, pH 9.0
Check the stock solution periodically for fluctuations in pH.
100 mM aminoallyl-dUTP
Dissolve 10 mg of aminoallyl-dUTP in 170 μl of ddH2O.
Add approximately 6.9 μl of 1 M NaOH to a final pH of 7.0
Check with litmus paper
50X aadUTP Stock Solution
8 μl of 100 mM aminoallyl-dUTP (Sigma)
10 μl of 100 mM dCTP
10 μl of 100 mM dGTP
2 μl of 100 mM dTTP
10 μl of 100 mM dATP
(See Hint #5 and Hint #6)
100 mM dTTP (Pharmacia)
100 mM dCTP (Pharmacia)
100 mM dGTP (Pharmacia)
10% SDS
100 mM dATP (Pharmacia)
SSC (20X)
pH 7.2
3 M NaCl
0.3 M Sodium Citrate
BioReagents and Chemicals
tRNA
poly A+ RNA
dCTP
aminoallyl-dUTP
dTTP
RNasin
Superscript II Reverse Transcriptase
dGTP
oligo-dT, anchored
dATP
Sodium Bicarbonate
SDS
DTT
Sodium Citrate
Sodium Hydroxide
Protocol Hints
1. The samples may be stored at -20°C indefinitely.
2. Monofunctional NHS-ester Cy3 (PA23001) and Cy5 dye (PA25001, Amersham) is supplied as a dry pellet. Each tube is sufficient to label ten reactions under normal conditions. Dissolve the dry pellet in 20 μl of Dimethyl Sulfoxide (DMSO). Aliquot 2 μl into ten single-use tubes, then dry under vacuum. Store in a dessicator at 4°C. 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.
3. Alternatively, dissolve the Cy dye in 10 μl of DMSO and add 1 μl of dye to 10.5 μl of the cDNA reaction. 10% DMSO in the coupling reaction will not affect the chemical reaction. Aliquot any unused dye and dry immediately.
4. An optional step is to reapply the flow-though for optimal binding.
5. This protocol uses a 4:1 ration of aminoallyl dUTP to dTTP. Altering the ratio of aminoallyl-dUTP will affect the incorporation of the Cy dye.
6. The 1X dNTP concentration during labeling is 500 μm each dATP, dCTP, dGTP, 400 μM aminoallyl-dUTP and 100 μm dTTP.
Citation and/or Web Resources
2. Slight modifications to original protocol by Mitch Garber and Anatoly Urisman.
1. Microarrays Homepage.
