Establishment And Evaluation Of Fluorescent Quantitative PCR For Detection Of Neutrophil Gelatinase Associated Lipocalin MRNA

Objective:Neutrophil gelatinase-associated lipocalin (NGAL), a novel member of lipocalin family, was found from neutrophil granulocyte by Kjeldsen et al in 1993. Growing evidence shows that NGAL is a protein with diversity of functions. NGAL has been implicated as a cell survival factor in many kinds of tumors. It is also a novel iron transporter with functions distinct from that of transferrion by binding a specific cellular receptor and mediate a new iron delivery pathway. And NGAL in the urine is an earlier marker than creatinine in serum in acute renal failure patients. So detection of NGAL has become more important than ever. In this study, we establish an SYBR green I based fluorescent quantitative PCR method for detecting mRNA levels of NGAL and validate it preliminarily in colorectal carcinoma and paracancerous tissue.Methods:1. PCR primers of NGAL were designed and synthesized. The amplified NGAL fragment was cloned into T vector. The recombinant plasmid was selected, identified, and was use as standard after its quantity had been determined.2. Fluorescent quantitative PCR primers were designed and synthesized according to the above-mentioned NGAL fragment .The optimization of the PCR reaction systems (such as concentration of primers and SYBR green I, annealing temperature) was determined by positive-cross test.3. The standard curve was prepared usingthe NGAL recombinant plasmid. After the FQ-PCR method was established, its linearity, specificity, sensitivity and reproducibility were evaluated.4.Twenty-six samples of colorectal carcinoma and paracancerous tissue were detected. And the results of FQ-PCR were compared with those of PCR.Results:1. Restriction maps showed that the structure of recombinant plasmid was the same as expected. 2.The optimized conditions of FQ-PCR are as follow: 2μl SYBR 10×Buffer (with Mg²⁺),0.2 mmol/L dNTPs,0.32μmol/ml primers,SYBR TaqTM 1.25μl,ddH2O15.15μl, Annealing temperature was 60.0℃.3.The standard curve obtained by plotting Ct values as a linear function of base 10 logarithm of the initial copies of template DNA. The correlation coefficient was 0.989. The inter-assay and intra-assay coefficient of variation were less than 4%. Not any nonspecific amplification was observed either. Compared with conventional PCR, FQ-PCR was 100-fold higher sensitive.4.The result shows that NGAL mRNA level in colorectal carcinoma tissue is significantly higher than that in paracancerous tissue. this new fluorescent quantitative method has many advantages than the PCR.Conclusions:The FQ-PCR method was established for quantitatively detecting NGAL mRNA. With rapid and sensitive property, which would lay a substantial foundation for detection of NGAL mRNA.quickly.