1. Mix 1-2 mg total RNA in nuclease-free
2 mg Oligo-dT. Add control RNA if necessary.
Adjust total volume to 13 ml with
If using mRNA, replace Oligo-dT with random primers.
Concentrating the RNA sample if necessary.
2. Heat to 70 °C for 5-10 minutes. Cool on ice for 5 minutes.
3. Add 12 ml of the following mixture:
4. Mix and incubate at 42 °C for 1-4 hours.
Hydrolysis and purification
5. Degrade RNA by addition of 15 ml of
0.1 N NaOH. Incubate at 70 °C for 10 minutes
6. Neutralize by addition of 15 ml 0.1 N HCl.
and purifying the labeled cDNA using Microcon-30 filter.
Warning: buffers containing free amine groups (e.g., Tris) interfer with the coupling
8. Concentrate to 10 ml.
9. Add 0.5 ml 1M sodium bicarbonate (pH 9.0).
10. Add 2 ml NHS-ester Cy3 or Cy5 dye.
11. Incubate at room temperature for 1 hour in the dark.
12. Quenching by adding 4.5 ml 4 M hydroxylamine.
13. Incubate at room temperature for 15 minutes in the dark.
and purifying the labeled cDNA using QIAquick or Microcon-30 filter.
15. OD measurements may be used to estimate the amount of cDNA and
incorporated Cy3/Cy5. You may need a cuvette with ≤ 10 ml
cDNA amount = A260 * 37 mg/ml * volume
Cy3 molecules/1000 bases = 58.5 * A550 / A260
Cy5 molecules/1000 bases = 35.1 * A650 / A260
Cy3 incorporated (pmols) = A550 * volume (ml) / 0.15
Cy3 incorporated (pmols) = A650 * volume (ml) / 0.25