One-step real-time fluorescence quantitative PCR for detecting WT1 mRNA expression in leukemia / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To establish a one-step real-time quantitative RT-PCR assay for detecting the expression of WT1 mRNA, which allows detection of the minimal residue disease and prognostic prediction in leukemic patients.</p><p><b>METHODS</b>WT1 gene fragment was amplified from the RNAs extracted from K562 cells using one-step RT-PCR. The quantitative standard were constructed by pMD 18-T vector cloning, and a Taqman-MGB fluorescent probe and a pair of primers were used to establish the one-step real-time fluorescence quantitative RT-PCR assay for WT1 gene detection. The sensitivity, repeatability and stability of this assay were evaluated and verified.</p><p><b>RESULTS</b>The sensitivity of this assay reached the 10(-4) level. The standard template of 1.0 x 10(6)-1.0 x 10(2) copies/ml were amplified by the one-step real-time fluorescence quantitative RT-PCR assay , and the Ct value was strongly correlated (r=0.998) to the logarithm of the initial template concentration. The repetition Ct value and both the inter-tube and inter-batch coefficients of variation (CV%) were less than 8%.</p><p><b>CONCLUSION</b>The one-step real-time fluorescence quantitative RT-PCR assay has good sensitivity, repeatability and specificity, and the one-step completion of the reverse transcription and PCR processes may reduce the operational complexities and the possibility of contamination.</p>