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 chain reaction (RT-PCR) to determine CK20 expression in colorectal tumor and hematopoietic tissue.


METHODS
Our method incorporates a calibrated PCR with an internal competitor and an external standard.


RESULTS
The RT-PCR assay is sensitive detecting 10 target molecules of CK20 in solution with one round of 38 amplification cycles. Genomic DNA contamination was eliminated by Dnase I digestion of total RNA. The inclusion of a calibrator in the quantitative RT-PCR analysis allowed for a high throughput of unknown samples within the same assay improving comparative analysis between the samples tested. Analysis of peripheral blood and bone marrow from 20 healthy volunteers revealed a low level of CK20 expression in all samples.


CONCLUSIONS
To study the clinical significance of CK20 expression as a marker of systemic metastatic disease it is essential to measure CK20 mRNA levels in hematopoietic tissue with sensitive quantitative RT-PCR. A sensitive and reproducible method, which is easily performed, is described.
van Eekelen
J A
JA
Department of Clinical Chemistry, Rogaland Central Hospital, Stavanger, Norway. piete-ga@online.no
Shammas
F V
FV
Wee
L
L
Heikkilä
R
R
Osland
A
A
D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics*; D001854:Bone Marrow Cells / Q000502:physiology; D002138:Calibration; D015179:Colorectal Neoplasms / Q000235:genetics; D004247:DNA / Q000032:analysis; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000235:genetics*; D053554:Keratin-20; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012235:Rhodamines / Q000378:metabolism; D012680:Sensitivity and Specificity
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D014408:Biomarkers, Tumor; D005456:Fluorescent Dyes; D007381:Intermediate Filament Proteins; C507499:KRT20 protein, human; D053554:Keratin-20; D012333:RNA, Messenger; D012235:Rhodamines; D004247:DNA