Application of quantitative RT-PCR using "TaqMan" technology to evaluate the expression of CK 18 mRNA in various cell lines.
Reverse transcriptase polymerase chain reaction (RT-PCR) is often used for sensitive detection of micrometastasis in peripheral blood, lymph nodes and bone marrow. While the utility of this method has been documented, it also has limitations in the detection of micrometastasis. The mRNA of target genes can be detected in healthy donors or in samples used for negative control, therefore the non-quantitativeness of conventional RT-PCR has been called into question. We analyzed the expression level of cytokeratin (CK) 18 mRNA in established esophageal and gastrointestinal carcinoma cell lines and non-epithelial cells, using quantitative RT-PCR, based on real time 'TaqMan TM' technology. CK 18 mRNA is more highly expressed in carcinoma cells than in non-epithelial cells. However, the expression level in non-epithelial cells was easily detected using conventional RT-PCR and agarose gel electrophoresis. In an analysis of CK 18 mRNA expression in peripheral venous blood in 13 healthy volunteers, we found that CK 18 mRNA was much less expressed than in cancer cell lines. However, the expression in all samples was at a level which was also detected using conventional RT-PCR. It would thus seem that not only qualitative, but also quantitative analysis, of the target mRNA is important to detect micrometastasis. Quantitative RT-PCR methods will make comparisons of the possible differences in expression levels of the target gene. For clinical applications, much further study is needed.
Tokunaga
E
E
Cancer Center, Kyushu University Hospital, Fukuoka, Japan.
Maehara
Y
Y
Oki
E
E
Koga
T
T
Kakeji
Y
Y
Sugimachi
K
K
D000328:Adult; D015179:Colorectal Neoplasms / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004938:Esophageal Neoplasms / Q000378:metabolism*; D005822:Genetic Vectors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007633:Keratins / Q000235:genetics*; D008175:Lung Neoplasms / Q000378:metabolism*; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism*; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D012333:RNA, Messenger; D007633:Keratins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases