Sensitive and quantitative one-step polymerase chain reaction using capillary electrophoresis and fluorescence detection for measuring cytokeratin 19expression

An improved quantitative assay to measure cytokeratin 19 (CK19) expression has been developed. The assay utilizes reverse transcription and a one-step polymerase chain reaction (PCR), with capillary electrophoresis and fluore scent labelling, to separate and detect the PCR products. Calibration curve s were constructed from a serial dilution of CK19 cDNA coamplified with a f ixed amount of CK19 internal standard, which was found to be linear between 10 and 500 molecules. Quantitative measurement of CK19 in samples was carr ied out by coamplifying the cDNA with a fixed amount of internal standard. The values were calculated from the calibration curve. The integrity of RNA and cDNA synthesis was checked by quantitative measurement of the breakpoi nt cluster region (BCR) gene expression. The assay is sensitive, detecting <10 CK19 transcripts, and reproducible with a coefficient of variation of a pproximately 10%. CK19 expression showed overlapping values when measured i n samples from peripheral blood and bone marrow in operable breast cancer p atients, in healthy volunteers or patients without epithelial cancer and in blood samples from patients with metastatic breast cancer. As the assay is easier to perform than traditional quantitative competitive PCR assays, it might be useful for quantitative measurement of other specific transcripts .