title: Immunobead RT-PCR versus regular RT-PCR amplification of CEA mRNA in peripheral blood.
abstract: OBJECTIVE
  The reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA has been used for the detection of cancer cells in peripheral blood. More recently, an immunomagnetic isolation and reverse transcription polymerase chain reaction (immunobead RT-PCR) was developed which has reportedly significant advantages over the previous RT-PCR analysis. In our study, we compared these two methods using a model set of peripheral blood containing tumor cells under standardized conditions.
  
  
  METHODS
  In order to compare the false positive rate, normal peripheral blood samples from five volunteers were analyzed by both methods. A model set of peripheral blood containing tumor cells was established by adding SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PCR products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobead RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic beads before the RT-PCR analysis.
  
  
  RESULTS
  The immunobead RT-PCR yielded no non-specific band, while the regular RT-PCR using total RNA did show non-specific band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobead RT-PCR allowed the detection of 10(1) tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 10(2) tumor cells in 1 ml of peripheral blood.
  
  
  CONCLUSIONS
  The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our model system.
authors.affiliation: Department of Biochemistry, Dongguk University School of Medicine, Kyung-Buk, Korea.
authors.forename: S
authors.initials: S
authors.lastname: Park
authors.forename: B
authors.initials: B
authors.lastname: Lee
authors.forename: I
authors.initials: I
authors.lastname: Kim
authors.forename: I
authors.initials: I
authors.lastname: Choi
authors.forename: K
authors.initials: K
authors.lastname: Hong
authors.forename: Y
authors.initials: Y
authors.lastname: Ryu
authors.forename: J
authors.initials: J
authors.lastname: Rhim
authors.forename: J
authors.initials: J
authors.lastname: Shin
authors.forename: S C
authors.initials: SC
authors.lastname: Park
authors.forename: H
authors.initials: H
authors.lastname: Chung
authors.forename: J
authors.initials: J
authors.lastname: Chung
mesh_terms: D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen / Q000097:blood / Q000235:genetics*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics / Q000276:immunology*; D012333:RNA, Messenger / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D012333:RNA, Messenger