1996 Fiscal Year Final Research Report Summary
Detection of ciculating prostate cencer cells with reverse-fromscriptabe polymelare chain reaction of PSA mRNA
| Project/Area Number |
07457366
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | Tohoku University |
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Principal Investigator |
ORIKASA Seiichi School of Medicine, Tohoku University, Professor, 医学部, 教授 (60001004)
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| Co-Investigator(Kenkyū-buntansha) |
FUNATO Tadao Tohoku University, Lecturer, 医学部・付属病院, 講師 (70165455)
HOSHI Senji Tohoku University, associate professor, 医学部, 助教授 (80107200)
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| Project Period (FY) |
1995 – 1996
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| Keywords | PSA mRNA / RT-PCR / Prostate Cancer / Staging |
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| Research Abstract |
In 1992 Morcno et al.introduced the concept of detecting circulating prostete cancer cells with reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of PSA mRNA.In 1994 Katz et al.correlated pathologicel stage and presence of circulating prostate cancer cells. Alimitation of these PCR assays is their complexity and subjective gel analysis.
We developed a rapid ultrasensitive PCR PSA detection method that replaced gel electrophoresis with colorimetric ELISA assay. The labor intensive Ficoll fractionation method was compared to a serum or plasma sample. Technique were compared with respect to cell count, total RNA and PSA message. Total RNA was extracted from LNCap, PC-3, DU-145, bladder cancer cell lines, 60 negative 10 BPH and 10 patients with advanced prostate cancer. We used rTth Polymerase for RT and Polymerase activity. The 226bp target was PCR- and clone-sequence verified as PSA only. The RNA target is amplified by RT-PCR with dinitrophenyl (DNP)-labelled primer. The PCR product is denatured and then is hybridized on a PSA secific probe-coated microwell plate. The DNA-probe hybrid is dectected colorimetrically using enzyme-antibody method.
The ELISA took 2 hours compared to 12 hours for Southen hybrydization. The ELISA molecular sensitivity was 60* more sensitive than ethidium bromide gel, eqivalent to digoxigenin. All cancers and 2 BPH tested were positive while all controls were negative.
The ELISA detection assay of PSA PCR products in an ultrasensitive that produces a grest information which is the first step in standarizing molecular staging assays. The determination of clinically significant circulating levels of PSA mRNA in men is required.
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