Microfluidic Digital-Quantitative PCR Analysis of MicroRNAs

Description:
The altered expression of microRNAs (miRNAs) is associated with various diseases. Extracellular miRNAs are found in almost all biological fluids such as blood and breath condensate and therefore have the potential to be used as diagnostic biomarkers. Reverse transcription-quantitative PCR (RT-qPCR) can quantify miRNAs, but is experimentally complex and expensive, making it prohibitive for routine miRNA analyses. Here, we developed a rapid one-step RT-free PCR assay to quantify miRNAs from biological fluids with limited sample preparation. This was achieved by translating base-stacking PCR into a bead-based microfluidic digital PCR (dPCR) assay. Primers were first designed to have optimal base-stacking energies with the miRNA let-7a. Detection probes for let-7a were then designed and conjugated to beads. A ratio of 100,000 probes per bead produced high dPCR signals in positive controls compared to negative controls, providing the optimal signal-to-noise ratio. Capture of let-7a onto beads was then optimized by varying sample incubation conditions. Upon validating miRNA analysis from buffer, we sought to demonstrate that let-7a can be measured in biological fluids. Let-7a was spiked into serum and breath condensate samples and captured onto beads. dPCR successfully quantified miRNA from both matrices with limits of detection <50 fM. These results show that base-stacking dPCR analyzes miRNA more quickly and less expensively that RT-qPCR, which demonstrates its potential for routine diagnostics.

Speaker: Brice Vanness - Wayne State University