Real-time fluorescence quantitative reverse transcription PCR in determination of guanylyl cyclase-C gene expression in peripheral blood of patients with colorectal cancer and its clinical significance / 第二军医大学学报

ABSTRACT

Objective:To develop a real-time fluorescent quantitative reverse transcription polymerase chain reaction(RFQ-RT-PCR) system for determination of the expression of guanylyl cyclase-C(GC-C) mRNA in the peripheral blood mononuclear cells(PBMC) in patients with colorectal cancer.Methods: The specific primers and TaqMan probe targeting the high conservative region of GC-C gene were designed for PCR amplification and the fluorescence was monitored in a real time manner.The expression levels of GC-C mRNA in clinical samples(including 30 healthy blood donors,11 patients with benign intestinal lesions and 37 with colorectal cancer) were calculated according to the standard curve.The mRNA levels of GC-C were presented as the ratios of GC-C mRNA to ?_2-microgluobulin mRNA.Results: The linear detection range of this RFQ-RT-PCR system was from 10~1 to 10~9 pg/ml and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 6.87% to 11.12% and from 8.86% to 15.19%,respectively.GC-C mRNA was expressed in 31/37 colorectal cancer patients,but not in healthy blood donors and patients with benign intestinal lesions.The mean level of GC-C mRNA was(0.88?)(0.06) in the above 31 colorectal cancer patients.GC-C mRNA levels were positively correlated to the Duke stage,lymph node metastasis and liver metastasis but not to patients' age,sex and tumor size.Conclusion: RFQ-RT-PCR has good sensitivity,specificity and reproducibility in determination of GC-C mRNA levels.Determination of GC-C mRNA levels in colorectal cancer patients may play a role in assessing Duke stage,lymph node metastasis,liver metastasis and approaching post-operation therapy.