posted on 2024-01-06, 14:04authored byBrice
C. Vanness, Thomas H. Linz
Proteins and microRNAs (miRNAs) act in tandem within
biological
pathways to regulate cellular functions, and their misregulation has
been correlated to numerous diseases. Because of their interconnectedness,
both miRNAs and proteins must be evaluated together to obtain accurate
insights into the molecular pathways of pathogenesis. However, few
analytical techniques can measure both classes of biomolecules in
parallel from a single biological sample. Here, microfluidic digital
quantitative PCR (dqPCR) was developed to simultaneously quantify
miRNA and protein targets in a multiplexed assay using a single detection
chemistry. This streamlined analysis was achieved by integrating base-stacking
PCR and immuno-PCR in a microfluidic array platform. Analyses of let-7a
(miRNA) and IL-6 (protein) were first optimized separately to identify
thermocycling and capture conditions amenable to both biomolecules.
Singleplex dqPCR studies exhibited the expected digital signals and
quantification cycles for both analytes over a range of concentrations.
Multiplexed analyses were then conducted to quantify both let-7a and
IL-6 with high sensitivity (LODs ∼ 3 fM) over a broad dynamic
range (5–5000 fM) using only standard PCR reagents. This multiplexed
dqPCR was then translated to the analysis of HEK293 cell lysate, where
endogenous let-7a and IL-6 were measured simultaneously without sample
purification or pretreatment. Collectively, these studies demonstrate
that the integration of BS-PCR and immuno-PCR achieves a sensitive
and streamlined approach for multiplexed analyses of miRNAs and proteins,
which will enable researchers to gain better insights into disease
pathogenesis in future applications.