Total RNA from hepatoma and the para-hepatoma liver tissues and the poly(A) RNA was prepared as described before [26, 27] . SSH was performed with the Clontech PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA) as described by the manufacturer but with the following modifications. Starting material consisted of 2 μg hepatoma mRNA as tester and 2 μg nonhepatoma liver tissue mRNA as driver, and vice versa. Primary and secondary PCR conditions were altered to increase specificity of amplification according to either plan A or B. Both A and B reduced the extension time and the number of cycles of the primary PCR to 2 min and 26 cycles, respectively. The primary PCR products were diluted 1/50 prior to use in the secondary PCR. All other aspects of plan A were as per the instructions of the manufacturer. Plan B diverged from plan A in two ways. First, the initial cycle of primary PCR was performed using annealing and extension times that had been reduced to 15 s and 1.5 min, respectively. Second, for subsequent cycles, the denaturing time was increased to 10 s while the annealing and extension times were reduced to 15 s and 1.5 min, respectively.