Clinical Chemistry Siemens Point of Care - Urinalysis
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Clinical Chemistry 43: 2244-2250, 1997;
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(Clinical Chemistry. 1997;43:2244-2250.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA

Peter Tschentscher, Christoph Wagenera and Michael Neumaier

Department of Clinical Chemistry, Medical Clinic, University Hospital Eppendorf, Martinistr. 52, D-20251 Hamburg, Germany.
a Author for correspondence. Fax 0049-40-4717-4621; e-mail wagener{at}uke

Processed pseudogenes of residual contaminating genomic DNA interfere with a sensitive detection of cytokeratin 18 (CK18) mRNA by reverse transcription and polymerase chain reaction (RT-PCR). This may cause false-positive results when CK18 mRNA is used as a marker for ectopic tumor cells in specimens from cancer patients. To establish a sensitive CK18 RT-PCR by excluding the amplification of processed pseudogenes, the following strategy was chosen: (a) CK18 pseudogene sequences were cloned from genomic DNA by PCR; (b) cDNA-specific primers were designed on the basis of mismatches between pseudogenes and cDNA; (c) PCR conditions were adjusted to reach maximum sensitivity and specificity. Epithelial cells (1–10) could be detected in 1 mL of blood. Among the numerous CK18 genes homologous to the transcribed gene, at least two different processed pseudogenes exist that are highly homologous to each other and to the exons of the transcribed CK18 gene.






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Copyright © 1997 by the American Association for Clinical Chemistry.