Figure 1

Evaluation of subtraction efficiency and the presence of potential differentially expressed genes in the subtracted libraries. To determine the subtraction efficiency, β-actin was PCR amplified using a primer set located within the very 3'-end of Rsa I digested β-actin fragment following the second round of hybridization. PCR products were electrophoresed on an agarose gel. No β-actin product was detected after 40 cycles of PCR amplification in a subtracted library, whereas β-actin was detected after 25 cycles of amplification in corresponding un-subtracted library (A). To amplify differentially expressed genes in circulating cells of healthy men and PCa patients, two rounds of PCR amplification was performed following hybridization steps described in Materials and Methods. To demonstrate the presence of potential differentially expressed genes in the subtracted libraries, the final PCR products were analyzed on a 1.5% agarose gel followed by ethidium bromide staining. We detected a series of distinct bands ranging from 300 to 1,000 bp. These DNA fragments represented genes that are either present (B, lane 1) or absent (B, lane 2) in circulating cells of PCa patients.