2006•Academic Journal of Second Military Medical UniversityRequires access
TaqMan real time quantitative RT-PCR in detection of peripheral blood CK19 mRNA
Jing Wei, BI Jian-wei, Jun Zhang, Wei Guo, Wenjie Li
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Abstract
Objective:To establish a quantitative method for determination of CK19 mRNA with TaqMan real time quantitative RT-PCR.Methods: A 230 bp fragment of CK19 mRNA was amplified from the total RNA of gastric cancer cells using RT-PCR methods and was introduced into pMD 18-T Simple vector.The plasmid was purified and the fluorescent standard PCR product was prepared.The expression levels of CK19 mRNA in standard PCR product,5 tumor tissue specimens and 30 healthy subjects were observed.Results: A 230 bp fragment of CK19 mRNA was successfully cloned into the pMD 18-T Simple vector and was verified by sequence analysis.A stable standard for detection of CK19 mRNA was established,that is,when C_(T) was set within 35 cycles,negative specimen was defined when the result was lower than 100 copies.Conclusion: TaqMan real time quantitative RT-PCR is stable and reliable in quantitative detection of CK19 mRNA in peripheral blood.