Dynabeads® Epithelial Enrich and Dynabeads® mRNA DIRECT
TM Micro Kit are particularly designed for epithelial cell enrichment and mRNA isolation from these enriched cells. This system makes use of magnetic separation technology to positively select rare tumour cells in whole blood or bone marrow samples for minimal residual cancer research. The presence of circulating tumour cells in the blood is a potential indicator for metastatic disease in most cancers including Prostate Cancer (PC) in which the detection of circulating Prostate Specific Antigen (PSA)-producing cells may identify patients who might benefit from early intervention.
I have used Dynabeads® Epithelial Enrich for positive selection of epithelial cells. These beads are coated with the monoclonal antibody Ber-EP4 against the human epithelial antigen (HEA), which expressed on human normal and malignant cells of epithelial origin. As a positive control, I spiked one hundred LNCaP (PSA positive) prostate cancer cells into a 5 ml anticoagulated normal blood sample before positive selection of epithelial cells. Then isolated cells were lysed using lysis/binding buffer and mRNA was extracted using Dynabeads® mRNA DIRECTTM Micro Kit, which is designed to isolate poly(A)+ RNA directly from small samples by using Dynabeads Oligo (dT)25. The company recommends to immediately using the mRNA-Dynabeads complex for RT-PCR but we eluted the mRNA from the beads for storage purposes and to be used in our RT-PCR system. Using specific primers for PSA mRNA, the one hundred spiked LNCaP cells gave a strong positive signal in a RT-PCR reaction.
Achieving an undetectable serum PSA concentration following radical prostatectomy does not guarantee the absence of residual cells and relapse might happen even when the serum PSA concentration is within normal range. This lack of correlation between serum PSA and patients at risk of relapse highlights the need for more sensitive alternative assays for residual disease detection. We believe the system we optimised for detection of residual PSA-producing prostate cells in peripheral blood is a very sensitive one and we are currently applying it on different groups of PC patients. To increase sensitivity of detection, I perform a nested PCR using inner primers for any negative samples from the first PCR and interesting results obtained.
The advantages of this system are its simplicity compared to others; reliability and high sensitivity due to cell enrichment before mRNA isolation. Compared to peripheral blood mononuclear cell (PBMC) preparation, the chance for detection of residual cells in whole blood is higher due to the possibility of epithelial cell loss by Ficoll separation. Most of RNA isolation kits from peripheral blood deal with relatively small volumes (0.5 to 1 ml) while with this system we isolate epithelial cells from larger volumes (5 ml) increasing the opportunity of detecting any tumour cells.
The only disadvantage, I can so far tell, is the expensive price for Dynabeads® Epithelial Enrich, which can be a limiting factor for screening many samples.
In summary, this system works efficiently with the maximum feasible sensitivity for detection of residual prostate cancer cells and it is possible to apply it for detection of any other circulating tumour cells from epithelial origin.
Eyad Elkord
Graduate Student
Department of Medical Biochemistry and Immunology
University of Wales, College of Medicine