Labeling of cDNA --- Cy3/Cy5 direct

RT

1. Mix 5-20 mg total RNA in nuclease-free ddH2 with 2 mg Oligo-dT. Add control RNA if necessary. Adjust total volume to 32 ml with nuclease-free ddH2.

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:

5X RT Buffer 10 ml
RNase Inhibitor 1 ml
dATP, dTTP, dGTP (25 mM each) 1 ml
1 mM dCTP 2 ml
1 mM Cy3-dCTP or Cy5-dCTP 2 ml
Superscript RT II 2 ml

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.

7. Dilute and purifying the labeled cDNA using QIAquick or Microcon-30 filter.

Coupling efficiency

8. OD measurements may be used to estimate the amount of cDNA and incorporated Cy3/Cy5. You may need a cuvette with ≤ 10 ml volume.

  • cDNA amount = A260 * 37 mg/ml * volume
  • Cy3 molecules/1000 bases = 58.5 * A550 / A260
  • Cy5 molecules/1000 bases = 35.1 * A650 / A260

    Or

  • Cy3 incorporated (pmols) = A550 * volume (ml) / 0.15
  • Cy3 incorporated (pmols) = A650 * volume (ml) / 0.25