Recived 6/10/97; Accepted 7/1/97

3. MATERIALS AND METHODS

Total RNA was extracted from 5 million K562 cells grown for three days in the absence and presence of nitric oxide as previously described. (5)

1. Duplicate cDNA synthesis reactions (Boehringer Mannheim 1st strand cDNA synthesis kit were performed using downstream primer 9 (T11GG) as the anchoring primer (Display Systems Differential Display kit, PGC Scientific, Gaithersburg, MD), according to the manufacturers instructions.

2. PCR was performed using the same anchoring primer and one of 4 different random upstream primers (10, 11, 12, and 13) supplied in the Display Systems kit. PCR was performed in a Perkin Elmer 9600 thermocycler using the following conditions: 94°C for 3 minutes, then 40 cycles of 94°C for 20 seconds, 40°C for 20 seconds, 72°C for 30 seconds followed by a final elongation at 72°C for 5 minutes. Trace amounts of ³⁵S-dATP was included in the PCR master mix per manufacturers protocols.

3. Amplified fragments from the two cDNA fractions were separated on a 6% denaturing acrylamide gel, transferred and dried onto Whatmann 3M paper and exposed to film for 10 days.

4. Differentially expressed bands were cut from a 6% denaturing acrylamide gel and eluted into 100 microliters of water by boiling for 15 minutes.

5. The eluted DNA samples were then used as templates for PCR amplification. 2.5 to 10 microliters of the eluted product was used in a 50 microliters PCR reaction containing 5 microliters 10x PCR buffer with 15mM MgCl₂(Boehringer Mannheim, Indiannapolis, IN) 5 microliters 500micromolar dNTPs (Boehringer Mannheim), 2 microliters 25 micromolar downstream primer 9 (Display Systems), 25 microliters 2 microM upstream primer 10 or 12 (Display Systems), and 0.5 microliters Taq polymerase (Boehringer Mannheim). Cycling conditions were identical to those used in step 2.

6. PCR products were electrophoresed in a 2% agarose gel and stained with ethidium bromide.