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Real-time reverse transcription loop-mediated isothermal amplification for rapid and simple quantification of WT1
Soji Morishita1, Hidenori Tani, Shinya Kurata
1Department of Life Science and Medical Bio-science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, Japan.
Objectives:
This study developed a novel MRD monitoring method targeting Wilms' tumor gene (WT1) mRNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Design And Methods:
A primer set for the assay was designed on the basis of the sequences between the 17AA and KTS regions of WT1mRNA. WT1 mRNA was quantified by real-time RT-LAMP and the accuracy of RT-LAMP was compared with that of real-time RT-PCR.
Results:
The standard curve was expressed as a linear relationship between the log copy numbers of WT1 mRNA ranging from 6.8 x 10 to 6.8 x 10(9) copies and the threshold time with a correlation coefficient of R(2) > 0.994. The measured values obtained by RT-LAMP strongly correlated with those obtained by real-time RT-PCR.
Conclusion:
RT-LAMP can be used to determine WT1 mRNA expression levels. This assay will contribute to a more specific, simple, and rapid MRD monitoring than conventional assays.
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