Fluorescent Probe Preparation

The following protocol describes how to make Cy3 and Cy5 labelled fluorescent probes from RNA samples using reverse transcription and an amino-allyl modified nucleotide for esterification of the Cyanine dyes. It is optimal for yeast RNA and may need modification with other RNAs.

Materials Required:

1-3 µg mRNA (or 15-20 µg Total RNA)
5 mg/ml oligo dT (5 µg)
40x aa-dUTP/dNTPs
SuperScript II & buffers
Monoreactive Cy dyes
Microcon 30 (2)
Qiagen PCR cleanup kit (1)
1M HEPES pH 7
0.5 M EDTA & 1N NaOH
42 & 65 °C heat sources

The first step is to reverse transcribe the RNA into cDNA in the presence of the amino-allyl nucleotide. Then we stop the reaction by base hydrolysis of the RNA. After neutralization and clean up the cDNA is allowed to conjugate to the reactive Cy dyes. Once that reaction is quenched and cleaned up, the probe is ready to go.

I. Reverse Transcription

Oligo dT or Random Prime RNA

	[   ]	µL
Oligo d(T)19N	2 mg/ml	1
pd(N)6 or 9	2 mg/ml	1
RNA (> 75 µg/ml)	1 to 3 µg	13.5
Total Volume		15.5

• Incubate at 70 °C for 10 min.
• Chill on ice
• add components of RT Mix, or 14.5 µl per reaction.

RT Mix	[   ]	µl
FSB	5x	6
aa-dUTP/dNTPs	40x	0.75
DTT	0.1M	3
SuperScript II	200 U/µl	1.9
Water		2.85

• incubate at 42 °C, 2 hrs.

II. Hydrolysis of RNA

Add:	1 µl	0.5M	EDTA
	5 µl	1N	NaOH
Incubate:	10 min. at 65 °C
Neutralize:	25 µl	1M	HEPES pH 7.0

III. Reaction Cleanup

It's important to remove unincorporated aa-dUTP before proceeding to the conjugation. A microcon 30 can be used to remove small molecules from the reaction. Also if Tris is used in the neutralization step it must be removed prior to proceeding. The amine groups on the Tris can react with the monofunctional NHS-ester of the Cy dye.

• place 450 µl H2O in Microcon 30
• add reaction
• spin at 12K RPM for 12 minutes
• remove flow-through
• Repeat wash 2X with H2O
• Invert microcon in a new tube and elute by spinning 1 minute.
• Can be dried down in spin vac and stored at -20 C

IV. Coupling of Cye dye    

• Resuspend cDNA in 4.5 µl of H2O
• Resuspend aliquot of Cy dye in 4.5 µl 0.1 M Carbonate Buffer, pH 8.5-9.0
• mix cDNA and Cy dye.
• incubate in the dark for 1 hr at Room Temp.

V. Quench Reaction

Quench any un-reacted Cy dye by adding primary amines.

• add 4.5 µl 4M Hydroxylamine.
• incubate 15 min. in the dark at RT.

VI. Reaction Cleanup II and Hyb Prep

Cy3 and Cy5 reactions can be combined and cleaned up together or seperately. Removal of free dye can be done with either gel filtration via spin column or pasteur pipette, or with various kits, such as Qiagen PCR cleanup kit.

The Qiagen PCR clean up kit works well for this step. However it is important to keep in mind the DNA binding curve for silica, on which this kit is based, is favorable at low pH but falls off precipitously around pH 8. Thus it is essential that the pH of your reaction be below 7.5 by the time it hits the Qiagen membrane. Alternatively, the reaction can be cleaned up using a protocol for Zymo columns, which has the benefit of very small elution volumes.

Protocol for Qiagen PCR clean-up kit:

• add 35 µl 100 mM NaOAc pH 5.2 to each reaction
• Combine reactions to 1 tube
• add 500 µl PB Buffer
• apply sample to QIAquick column
• spin 30-60 sec, ~13,000 RPM (> 10,000 x g)
• discard flow-through, add 750 µl PE Buffer
• spin 30-60 sec
• discard flow-through
• spin 1 min.
• place column in new tube
• add 50 µl EB Buffer or H2O to membrane
• spin 1 min.

Eluted volume is typically around 48 µl (15.5 ul or less for Zymo Protocol). I elute with 1/2x PE buffer and reduce the volume for hybridization in the spin vac.

You can check your probe by scanning it in a spectrophotometer (200-700nM). You should see three peaks of absorbance. One at 260 nM for the cDNA, and then one each at 550 and 650 nM for Cy3 and Cy5 respectively.

dNTP Mix

dNTP	1x	40x	Stock [ ]	Volume
dATP	500 µM	20 mM	100 mM	10 µL
dCTP				10 µL
dGTP				10 µL
dTTP	300 µM	12 mM	100 mM	6 µL
aa-dUTP	200 µM	8 mM	50 mM	8 µL
H2O				6 µl
Total				50 µl

Labelling Materials

dNTP Calculator - Calculates the concentration of aa-dUTP when the absorbance is entered.