Biotech Glossary |
Bioinformatics |
Lab Protocol |
Notes |
Malaysia University |
Amino-allyl Reverse Transcription
Overview
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. In this protocol, cDNA is labeled with Cy3- and Cy5-fluorescent dyes so that the probes can be detected when hybridized to the microarray.
Procedure
A. Preparation of Cy dyes
1. Resuspend the contents of one pack of the monofunctional NHS-ester Cy3 or Cy5 dye in 72 μl of Dimethyl Sulfoxide (DMSO).
2. Aliquot 4.5 μl per tube for a total of sixteen tubes.
3. Use these aliquots immediately or lyophilize by dessicating under vacuum.
4. The dried aliquots can be stored at 4°C in a dessicator.
5. Use one aliquot of the dyes for each Reverse Transcriptase labeling reaction and microarray hybridization.
B. Reverse Transcription
1. Combine the following:
1 μl of oligo dT/pdN6
1 to 4 μg of polyA+ RNA
Adjust volume to a total of 15.5 μl
2. Incubate the mixture at 70°C for 10 min.
3. Chill on ice for 10 min.
4. Add the following for the cDNA synthesis:
6 μl of 5X Buffer
0.6 μl of 50X aa-dUTP/dNTPs
3 μl of 0.1 M DTT
1.9 μl of Superscript II Reverse Transcriptase
3 μl of ddH2O
4. Incubate at 42°C for 2 hr
C. Hydrolysis
1. Add 10 μl of 1 M NaOH.
2. Add 10 μl of 0.5 M EDTA.
3. Incubate for 15 min at 65°C.
4. Neutralize by adding 25 μl of 1 M Tris, pH 7.4 or 1 M HEPES, pH 7.5.
D. Clean-up
To continue with the amino-allyl dye coupling procedure, all Tris must be removed from the reaction to prevent the monofunctional NHS-ester Cye dyes from coupling to free amine groups in solution.
1. Fill one Microcon 30™ concentrator with 450 μl of ddH2O.
2. Add the neutralized reaction.
3. Spin at 12,000 X g for 8 min.
4. Discard the flow-through.
5. Repeat the process twice by refilling and centrifuging the original filter.
6. Elute into a fresh tube.
7. Lyophilize the eluate under vacuum.
E. Coupling
1. Resuspend the cDNA pellet in 9 μl of 0.1 M Sodium Bicarbonate Buffer.
2. Add the suspended cDNA solution to the dried aliquot of Cy3 or Cy5 dye.
3. Mix the dye and the cDNA.
4. Incubate for 1 hr at room temperature in the dark.
F. Quenching and Clean-up
Before combining the Cy3 and Cy5 samples for hybridization, the reactions must be quenched to prevent cross-coupling.
1. Add 4.5 μl of 4 M Hydroxylamine.
2. Incubate the reaction for 15 min at room temperature in the dark.
3. To remove any unincorporated/quenched Cy dyes, proceed with Qia-Quick™ PCR purification kit.
4. Combine the Cy3 and Cy5 reactions.
5. Add 70 μl of ddH2O.
6. Add 500 μl of Buffer PB (Supplied in the Qia-Quick™ PCR purification kit).
7. Apply to Qia-Quick™ column and centrifuge at 10,000 X g for 30 to 60 sec.
8. Aspirate away the flow-through.
9. Add 750 μl of Buffer PE (supplied in the Qia-Quick™ PCR purification kit) and centrifuge at 10,000 X g for 30 to 60 sec.
10. Aspirate away the flow-through. Repeat the process twice more.
11. Aspirate the flow-through and centrifuge for 1 min at high speed to dry the column.
12. Transfer to a fresh microcentrifuge tube.
13.Add 30 μl of Buffer EB (supplied in the Qia-Quick™ PCR purification kit) to the center of the filter.
14. Incubate 1 min at room temperature.
15. Centrifuge at 12,000 X g for 1 min.
16. Elute again.
G. Hybridization Preparation
1.Lyophilize the Qia-Quick™ eluate under vacuum.
2. Adjust the volume to a total of 18 μl with ddH2O and Hepes so that the final concentration of Hepes is 25 mM.
3. Add 3.6 μl of 20X SSC.
4. Add 0.54 μl of 10% SDS.
5. Incubate the reaction at 100°C for 2 min.
6. Apply to the prepared microarray.
Solutions
Superscript II Reverse Transcriptase
0.1 M DTT
polyA+ RNA
10mg/ml poly+ RNA
0.1 M Sodium Bicarbonate Buffer
Adjust pH to 9.0
50X aa-dUTP/dNTPs
4 μl of 100 mM aa-dUTP
10 μl of dCTP
10 μl of dGTP
6 μl of 100 mM dTTP
10 μl of dATP
dNTPS (1X)
500 μM dATP
500 μM dCTP
200 μM aadUTP (5-(3-Aminoallyl)-2'-deoxyuridine 5'-triphosphate sodium salt, Sigma)
500 μM dGTP
300 μM dTTP
Oligo dT/pdN6
5 μg/μl Oligo dT/pdN6
DMSO
10% SDS
NHS-ester Cy5 dye
Amersham PA 25001
Mono-functional Cy5 reactive pack
20X SSC
3.0 M NaCl
300 mM Sodium Citrate (pH 8.0)
0.75 μl of 10% SDS
NHS-ester Cy3
Amersham PA 23001
Mono-functional Cy3 reactive pack
Qia-Quick™ PCR purification kit
4 M Hydroxylamine
CAUTION! See Hint #1
1 M HEPES, pH 7.5
1 M Tris, pH 7.4
0.5 M EDTA
5X Buffer for Superscript II Reverse Transcriptase
BioReagents and Chemicals
dCTP
dTTP
dGTP
dATP
polyA+ RNA
Reverse Transcriptase, Superscript II
DMSO
Hydroxylamine
Sodium Bicarbonate
DTT
Tris
Sodium Chloride
aadUTP
SDS
EDTA
Oligo dT/pdN6
Sodium Citrate
NHS-ester Cy5 dye
HEPES
NHS-ester Cy3
Dimethyl Sulfoxide
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
Citation and/or Web Resources
1.Lab website
