Quantitative analysis of cytokeratin 20 gene expression using RT-PCR and capillary electrophoresis with fluorescent DNA detection

Objective: We developed a quantitative reverse-transcription polymerase cha in reaction (RT-PGR) to determine CK20 expression in colorectal tumor and h ematopoietic tissue. Design and Methods: Our method incorporates a calibrated PCR with an intern al competitor and an external standard. Results: The RT-PCR assay is sensitive detecting 10 target molecules of CK2 0 in solution with one round of 38 amplification cycles. Genomic DNA contam ination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high through put of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow f rom 20 healthy volunteers revealed a low level of CK20 expression in all sa mples. Conclusion: To study the clinical significance of CK20 expression as a mark er of systemic metastatic disease it is essential to measure CK20 mRNA leve ls in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described. Copyright (C) 2000 The Canadian Society of Clinical Chemists.