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FLUORESCENT PROBE PREPARATION FROM RNA FOR DNA MICROARRAYS
Fluorescent Probe Preparation from RNA for DNA Microarrays
Contributor:
Chris Seidel
Overview
The following protocol describes a process for making Cy3- and Cy5-labeled fluorescent DNA probes from RNA samples, using reverse transcription and an amino-allyl modified nucleotide for esterification of the Cyanine dyes. It is optimized for yeast RNA and may need modification for use with other RNA from other sources.
Procedure
A. Reverse Transcription of RNA
The first step is to reverse transcribe the RNA into cDNA in the presence of the amino-allyl nucleotide. Stop the reaction by base hydrolysis of the RNA. 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. Add the following components to a 0.5 μl microcentrifuge tube:
Oligonucleoride dT Primed RNA
1 μl of Oligonucleotide dT
14.5 μg of Isolated RNA
--OR--
Random Primed RNA
1 μl of pd(N)6
14.5 μg of Isolated RNA
2. Incubate at 70°C for 10 min.
3. Chill the reaction on wet ice.
4. Add the following to each microcentrifuge tube:
6 μl of 5X FSB (1X final concentration)
0.6 μl of 50X aa-dUTP/dNTPs
3 μl of 100 mM DTT
1.9 μl of SuperScript II™ (BRL)
3 μl of ddH2O
5. Incubate at 42°C for 2 hr.
B. Hydrolysis of the RNA
1. Add the following to each microcentrifuge tube:
10 μl of 1 M NaOH
10 μl of 0.5 M EDTA
2. Incubate for 15 min at 65°C
3. Quench the reaction by adding to each microcentrifuge tube either:
25 μl of 1 M Tris, pH 7.4
--OR--
25 μl of 1 M HEPES, pH 7.0
C. Reaction Cleanup
It is important to remove unincorporated aa-dUTP before proceeding to the conjugation. A Microcon 30™ (Millipore) can be used to remove small molecules from the reaction. Also, if Tris is used in the neutralization step, it must be removed before proceeding to the conjugation reaction. The amine groups on the Tris moiety can react with the monofunctional NHS-Ester of the Cy Dye.
1. Add 450 μl of ddH2O to a Microcon 30™
2. Add the quenched reaction solution (prepared in Step #B3) to the Microcon 30™.
3. Centrifuge at 12,000 rpm (setting "12") in a microcentrifuge for 12 min at room temperature.
4. Remove the flow-through from the Microcon 30™.
5. Add 500 μl of ddH2O to the Microcon 30™ well.
6. Centrifuge at 12,000 rpm (setting "12") in a microcentrifuge for 12 min at room temperature.
7. Repeat Steps #C4 to #C6 one more time.
8. Invert the Microcon 30™ and elute the retentate into the flow-through reservoir (as per the Manufacture's instruction) by microcentrifuging for 1 min at 12,000 rpm (setting "12").
9. OPTIONAL: Dry the cDNA Solution completely in a vacuum centrifuge and store at -20°C.
D. Coupling of the Cye Dye
For each cDNA sample, Cy3 and Cy5 dyes should be prepared. The reactions for each dye need to be performed separately. The Cy3 and Cy5 dyes can be combined just before analysis (Section G).
1. Resuspend the cDNA Solution in 4.5 μl of ddH2O.
2. Resuspend an aliquot of Cy3 and Cy5 dyes in 4.5 μl of 0.1 M Carbonate Buffer.
3. Add the resuspended Cy Dye to the cDNA Solution.
4. Mix the solution well by flicking the side of the microcentrifuge tube.
5. Incubate the reaction in the dark for 1 hr at room temperature.
E. Quenching the Cye Dye Reaction
The unreacted Cye Dye can be neutralized with primary amines
1. Add 4.5 μl of 4 M Hydroxylamine to each reaction mixture.
2. Incubate the reaction in the dark for 15 min at room temperature.
F. Reaction Cleanup and Hyb Prep
Free dye can be removed by gel filtration with a centrifuge column or Pasteur pipette, or with various kits, such as Qiagen QIAquick® PCR cleanup kit. The Qiagen QIAquick® PCR cleanup kit works well. The DNA binding curve for silica is favorable at low pH but falls off precipitously around pH 8. Thus, it is essential that the pH of the reaction be below 7.5 before application to the QIAquick® membrane.
1. Add 35 μl of 100 mM NaOAc, pH 5.2 to each reaction mixture.
2. Combine the Cy3 and Cy5 reactions in a single microcentrifuge tube.
3. Add 500 μl of PB Buffer and mix well.
4. Apply the sample to a QIAquick® column.
5. Centrifuge in a microcentrifuge at approximately 13,000 rpm (setting "13" and greater than 10,000 X g) for 30 to 60 sec.
6. Discard the flow-through.
7. Add 750 μl of PE Buffer to the QIAquick® column.
8. Centrifuge in a microcentrifuge at approximately 13,000 rpm (setting "13" and greater than 10,000 X g) for 30 to 60 sec.
9. Discard the flow-through.
10. Centrifuge in a microcentrifuge at approximately 13,000 rpm (setting "13" and greater than 10,000 X g) for 1 min.
12. Transfer the QIAquick® column to a fresh microcentrifuge tube.
13. Add 50 μl of ddH2O to the QIAquick® column (see Hint #1).
14. Centrifuge in a microcentrifuge at approximately 13,000 rpm (setting "13" and greater than 10,000 X g) for 1 min.
15. Combine the Cy3 and Cy5 probes (see Hint #2).
16. Determine the cDNA probe intensity (Section G).
17. Dry the eluted solution in a vacuum centrifuge.
18. Resuspend the dried solution in 18 μl of ddH2O and use in Protocol ID#2217.
G. Determination of Probe Intensity
1. Add the Cy3 and Cy5 combined probe solution to a micro-volume Quartz cuvette.
2. Scan the solution from 200 nM to 700 nM.
3. Compare the scan with representative scans shown in Image #1.
4. Three peaks should be apparent: Peak #1 at 260 nM for the cDNA, Peak #2 at 550 for the Cy3 probe and Peak #3 at 650 nM for the Cy5 probe (see Hint #3).
5. Return to Step #F17.
Solutions
aa-dUTP/dNTPs (50X)
10 mM Amino-Allyl dUTP (see Hint #3)
25 mM dGTP
25 mM dATP
15 mM dTTP
25 mM dCTP 
FSB (5X)
Provided with SuperScript II™ (BRL)
5X First Strand Synthesis Buffer (FSB)
0.5 M EDTA
Isolated RNA
15 to 20 μg of Total RNA
Or
1 to 2 μg of mRNA
Isolate RNA from Yeast Cells (see Protocol ID#403)
PB Buffer
Provided with QIAquick® (QIAGEN)
pd(N)6
5 mg/ml Oligonucleotide containing six random deoxynuclotides
100 mM NaOAc, pH 5.2
100 mM Sodium Acetate, pH 5.2
Oligonucleotide dT
5 mg/ml Oligonucleotide dT
Cy5 Dye Aliquots
Dry in a vacuum centrifuge
Store desiccated in the dark at 2 to 8 *dec*C until use
Resuspend Cy5 Mono-Reactive Dye Pack (Amersham/Pharmacia) in 72 μl of DMSO (see Hint #4)
Aliquot 4.5 μl into 16 clean microcentrifuge tubes
Cy3 Dye Aliquots
Dry in a vacuum centrifuge
Store desiccated in the dark at 2 to 8 *dec*C until use
Resuspend Cy3 Mono-Reactive Dye Pack (Amersham/Pharmacia) in 72 μl of DMSO (see Hint #4)
Aliquot 4.5 μl into 16 clean microcentrifuge tubes
4 M Hydroxylamine
0.1 M Carbonate Buffer
0.1 M Sodium Carbonate, pH 8.5 to 9.0
1 M HEPES, pH 7.0
1 M Tris, pH 7.4
100 mM DTT
1 M NaOH
BioReagents and Chemicals
5-(3-Aminoallyl)-2'-Deoxyuridine 5'-Triphosphate
Sodium Hydroxide
Sodium Carbonate
DTT
Oligonucleotide
EDTA
DMSO
Tris
dCTP
HEPES
dTTP
dGTP
dATP
Cy5 Mono-Reactive Dye Pack
Sodium Acetate
Cy3 Mono-Reactive Dye Pack
Hydroxylamine
Protocol Hints
1. To elute the DNA from the QIAquick® column, EB Buffer can be substituted for ddH2O. The contributor of this protocol uses 0.5X PE Buffer for elution and then reduces the final volume by vacuum centrifugation.
2. The eluted volume from the QIAquick® column is approximately 48 μl per Cy reaction.
3. The extinction coefficients for 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (Amino-Allyl dUTP) in 0.1 M PO4, pH 7.5 are 7.1 mM at 289 nm and 10.7 mM at 249 nm.
4. Although DMSO takes a long time to vacuum concentrate, Cy5 is not soluble in the alternative, Dioxane. The contributor does not suggest using ddH2O since the mono reactive ester is labile in water.
