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)
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

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

II. Hydrolysis of RNA

Add: 1 µl 0.5M EDTA
5 µl 1N NaOH
Incubate: 10 min. at 65 °C
Neutralize: 25 µl1MHEPES 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.

IV. Coupling of Cye dye     notes

V. Quench Reaction

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

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:

Eluted volume is typically around 48 µl. 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.

Combined reactions should be in a volume of 18 µl (can use spin vac to control volume or dry down and resuspend in H2O). The precise colume you need will depend on the size of your cover slip - adjust volumes acordingly.

add: 3.6 µl 20X SSC
1.8 µl polyA (10 mg/ml)
0.54 µl 10% SDS
denature: 2 min. at 95 °C

The probe is now ready for slide hybridization.

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

Description Supplier Catalog # Price Notes
5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate Ambion 8439 50ul/50mM Soln, $100 This is the best deal.
5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate Sigma A0410 going up and up Forget Sigma, buy Ambion.
Cy3 + Cy5 Mono-Reactive dye pack Amersham RPN5661 $248.00 12 tubes each Cy3 & Cy5. Each tube can be used for 1-3 rxns. No aliquoting necessary.
Cy3 Mono-Reactive dye pack Amersham PA23001 $170.00 5 tubes, 12-16 rxns each.
Cy5 Mono-Reactive dye pack Amersham PA25001 $170.00 5 tubes, 12-16 rxns each.
Hydroxylamine (50% wt soln in H20) FW 33.03
(also available as HCl salt from Sigma H9876 100g/$10.40)
Aldrich 46,780-4 $15.50

Prepare aliquots* of Cy dyes as follows:

notes: These mono reactive esters are labile in water, and susceptible to moisture. DMSO is used as an aliquoting reagent to avoid this problem, but stability of the aliquots can still be a problem. This aliquoting method is only used for the larger dye packs (PA23001 & PA25001). Only make aliquots if you don't plan on using an entire tube all at once (i.e. 12-16 rxns). Otherwise it's better to use the smaller dye packs (RPN5661) and avoid making aliquots altogether.

The extinction co-efficient for the amino-allyl dUTP in 0.1 M PO4, pH 7.5 is:
Wavelength 289nm 249nm
Ext (mM) 7.1 10.7

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

This protocol came to me through Holly Bennet and Joe DeRisi and apparently was originated at Rosetta Informatics.
Chris Seidel Fall 1999 (revised Winter 2003)