DNA was extracted from frozen formalin-fixed melanoma primary tumor and lymph node sections by 40 min of incubation in lysis buffer [25 mM NaOH and 0.2 mM EDTA (pH 12)] at 95°C, followed by neutralization with equal volumes of neutralization buffer [40 mM tris-HCl (pH 5)]. RFP primers were as follows: 5′-CAGTTCCAGTACGGCTCCAAG-3′ (forward) and 5′- CCTCGGGGTACATCCGCTC-3′ (reverse). Actin primers were as follows: 5′-GAAGTACCCCATTGAACATGGC-3′ (forward) and 5′-GACACCGTCCC-CAGAATCC-3′ (reverse). Reactions were run with a 60°C annealing temperature.

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