PURPOSE: Reverse transcriptase-polymerase chain reaction (RT-PCR) offers a potentially more sensitive assay for detecting cells expressing prostate-specific antigen (PSA) mRNA in peripheral circulation. But the sensitivity and specificity are variable depending on the position of the PSA amplification. To increase sensitivity and specificity, the whole PSA cDNA (1466 bp) was separated into eight different parts. METHODS: We examined RT-PCR on 12 urogenital cell lines, including three prostate cancer (LNCaP, PC3, DU145), five human renal cell carcinoma (SMKT-R3, TOS-1, TOS-2, R4, ACHN), two urinary bladder cancer (YTS-1, KK-47) and two testicular cancer (NEC8, NEC14) cell lines. The sizes of the eight fragmented PSA used in the experiment were PSA-1 (1-257bp), PSA-2 (1-322bp), PSA-3 ( 172-507bp), PSA-4 (172-851 bp), PSA-5 (595-1347 bp), PSA-6 (682 967 bp), PSA-7 (682-1347 bp) and PSA-8 (863-1466 bp). RESULTS: All cell lines had positive signals from PSA-6, PSA-7 and PSA-8. The positive signals from PSA-1, PSA-2 and PSA-3 were detected in some other cell lines in addition to the three prostate cancer cell lines. Only LNCaP which produces the PSA protein had a positive signal from PSA-5. PC3 and DU145 (which do not produce PSA) and LNCaP had a positive signal from PSA-4. Therefore, the inner primer PSA-4' (578-782 bp) used to increase sensitivity and specificity. Nested RT-PCR on the 12 cell lines, using the PSA-4 and 4' primers, detected more clear bands in the three prostate cancer cells. CONCLUSION: Nested RT-PCR using PSA-4 (outer primer) and PSA-4' (inner primer) may be useful for detecting prostate cancer cells in the peripheral blood.
PURPOSE: Reverse transcriptase-polymerase chain reaction (RT-PCR) offers a potentially more sensitive assay for detecting cells expressing prostate-specific antigen (PSA) mRNA in peripheral circulation. But the sensitivity and specificity are variable depending on the position of the PSA amplification. To increase sensitivity and specificity, the whole PSA cDNA (1466 bp) was separated into eight different parts. METHODS: We examined RT-PCR on 12 urogenital cell lines, including three prostate cancer (LNCaP, PC3, DU145), five humanrenal cell carcinoma (SMKT-R3, TOS-1, TOS-2, R4, ACHN), two urinary bladder cancer (YTS-1, KK-47) and two testicular cancer (NEC8, NEC14) cell lines. The sizes of the eight fragmented PSA used in the experiment were PSA-1 (1-257bp), PSA-2 (1-322bp), PSA-3 ( 172-507bp), PSA-4 (172-851 bp), PSA-5 (595-1347 bp), PSA-6 (682 967 bp), PSA-7 (682-1347 bp) and PSA-8 (863-1466 bp). RESULTS: All cell lines had positive signals from PSA-6, PSA-7 and PSA-8. The positive signals from PSA-1, PSA-2 and PSA-3 were detected in some other cell lines in addition to the three prostate cancer cell lines. Only LNCaP which produces the PSA protein had a positive signal from PSA-5. PC3 and DU145 (which do not produce PSA) and LNCaP had a positive signal from PSA-4. Therefore, the inner primer PSA-4' (578-782 bp) used to increase sensitivity and specificity. Nested RT-PCR on the 12 cell lines, using the PSA-4 and 4' primers, detected more clear bands in the three prostate cancer cells. CONCLUSION: Nested RT-PCR using PSA-4 (outer primer) and PSA-4' (inner primer) may be useful for detecting prostate cancer cells in the peripheral blood.