1. Mix 5-20 mg total RNA in nuclease-free
2 mg Oligo-dT. Add control RNA if necessary.
Adjust total volume to 32 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 18 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
20 ml of 1 M Tris-HCl pH8.0.
and purifying the labeled cDNA using QIAquick or Microcon-30 filter.
8. 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