Our solution

To further increase the accuracy of our method, it is important to use the necessary calibrators, to correctly quantify the selected microRNAs. For this reason, we need a negative control sample, to determine the fluorescence that corresponds to the cell-free system or other components of the reaction. This sample should contain the reagents of the in vitro protein synthesis protocol and the toehold switch for each miRNA and the reagents of EXPAR reaction without the miRNA. Furthermore, we need three positive control samples for each miRNA. This sample should consist of all the necessary reagents for the reaction, plus the miRNA. Each positive control sample should contain the miRNA in a different known quantity. We need a sample with a very low quantity of the miRNA, one with an average quantity and one with the highest quantity of miRNA that produces a detectable change in the fluorescent signal.

The exact quantities of miRNAs for these control samples should be determined through clinical trials, based on the measurements of fluorescence and the concentration of miRNAs in the urine of patients with PDAC. Although we understand that this is a critical step for the function of our method, we have decided not to run these experiments, due to safety and bioethical issues. However, we hope that in the future we will be able to test our toeholds with real urine samples.

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Project Description

Who will use it?

Improving affordability: Choosing the appropriate amplification method

Moving towards a POC diagnostic

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