Sample LABELING by Reverse Transcription

1. If using an anchored oligo dT primer, anneal the primer to the RNA in the following 17 µl reaction (use a 0.2 ml thinwall PCR tube so that incubations can be carried out in a PCR cycler):

If using an oligo dT(12-18) primer, anneal the primer to the RNA in the following 17 µl reaction:

The incorporation rate for Cy5-dUTP is less than that of Cy3-dUTP, so more RNA is labeled to achieve more equivalent signal from each species.

2. Heat to 65°C for 10 minutes and cool on ice for 2 minutes.

3. Add 23 µl of reaction mixture containing either Cy5-dUTP or Cy3-dUTP nucleotides, mix well by pipetting and use a brief centrifuge spin to concentrate in the bottom of the tube:

Superscript polymerase is very sensitive to denaturation at air/liquid interfaces, so be very careful to suppress foaming in all handling of this reaction.

4. Incubate at 42°C for 30 min. then add 2 µl Superscript II. Make sure the enzyme is well mixed in the reaction volume and incubate at 42O C for 30-60 min.

5. Add 5 µl of 0.5M EDTA.

Be sure you have stopped your reaction with EDTA before adding NaOH, since nucleic acids precipitate in alkaline magnesium solutions.

6. Add 10 µl 1N NaOH, incubate at 65O C for 60 minutes to hydrolyze residual RNA. Cool to room temperature.

The purity of the sodium hydroxide solution used in this step is crucial. Slight contamination or long storage in a glass vessel can produce a solution that will degrade the Cy5 dye molecule, turning the solution yellow. Some researchers achieve better results by reducing the time of hydrolysis to 30 minutes.

7. Neutralize by adding 25 µl of 1M Tris-HCl (pH 7.5).

8. Desalt the labeled cDNA by adding the neutralized reaction, 400 µl of TE pH 7.5 and 20 µg of human C0t-1 DNA to a MicroCon 100 cartridge. Pipette to mix, spin for 10 minutes at 500 x g.

9. Wash again by adding 200 µl TE pH 7.5 and concentrating to about 20-30 µl (approximately 8-10 min at 500 x g).

Alternatively, a smaller pore MicroCon 30 can be used to speed the concentration step. In this case, centrifuge the first wash for approximately 4.5 minutes at 16,000 xg and the second (200 µl wash) for about 2.5 minutes at 16,000 xg.

10. Recover by inverting the concentrator over a clean collection tube and spinning for 3 min at 500 x g.

In some cases, the cy5 labeled cDNA will form a gelatinous blue precipitate that is recovered in the concentrated volume. The presence of this material signals the presence of contaminants. The more extreme the contamination, the greater the fraction of cDNA which will be captured in this gel. Even if heat solubilized, this material tends to produce uniform, non-specific binding to the DNA targets.

When concentrating by centrifugal filtration, the times required to achieve the desired final volume are variable. Overly long spins can remove nearly all the water from the solution being filtered. When fluor-tagged nucleic acids are concentrated onto the filter in this fashion, they are very hard to remove, so it is necessary to approach the desired volume by conservative approximations of the required spin times. If control of volumes proves difficult, the final concentration can be achieved by evaporating liquid in the speed-vac. Vacuum evaporation, if not to dryness, does not degrade the performance of the labeled cDNA.

11. Take a 2-3 µl aliquot of the Cy5 labeled cDNA for analysis, leaving 18-28 µl for hybridization.

12. Run this probe on a 2% agarose gel (6cm wide x 8.5 cm long, 2 mm wide teeth) in Tris Acetate Electrophoresis Buffer (TAE).

For maximal sensitivity when running samples on a gel for fluor analysis, use loading buffer with minimal dye and do not add ethidium bromide to the gel or running buffer.

13. Scan the gel on a Molecular Dynamics Storm fluorescence scanner (setting: red fluorescence, 200 micron resolution, 1000 volts on PMT)

Successful labeling produces a dense smear of probe from 400 bp to >1000 bp, with little pile-up of low molecular weight transcripts (as in Figure 1, Lane A). Weak labeling and significant levels of low molecular weight material indicates a poor labeling (as in Figure 1, Lane B). A fraction of the observed low molecular weight material is unincorporated fluor nucleotide.