Multiplexed miRNA and Protein Analysis Using Digital Quantitative PCR in Microwell Arrays

Abstract

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.

Figure 1.

(A) Photograph of a microwell array device. Dime shown for size comparison. The first inset shows a SEM image (3000X magnification) of a loaded device. The hexagonal pattern enables dense microwell packing. The second inset shows a SEM image (27000X magnification) of a bead loaded into a microwell. Microwells were engineered to only contain a single bead. (B) Fluorescence images of the same region of a microfluidic device at cycle 1 and cycle 25. A significant increase in fluorescence is observed in active microwells after 25 PCR cycles.

Figure 2.

The optimal guide number for let-7a capture beads was assessed by comparing digital signals from let-7a positive controls (100 fM, orange squares) and negative controls (0 fM, gray triangles) (n=3). Low guide numbers exhibit low signal due to inefficient capture and amplification. At 10,000 guides/bead, the positive control exhibits a high response that then plateaus at higher guide numbers. Non-specific amplification in the negative controls increases with increasing guide numbers across the range.

Figure 3.

The quantitative response of dqPCR was evaluated using let-7a concentrations of 0–5000 fM and a capture time of 2 h (n=3). Digital PCR signals (orange circles) exhibited a concentration-dependent increase with increasing concentrations, and a LOD of 1.5 fM. Quantitative PCR Cq values (black diamonds) shifted earlier with increasing concentrations because more copies of miRNA were initially present per microwell. The ability to use both digital and quantitative PCR dimensions expands the dynamic range of the assay.

Figure 4.

Detection antibody concentration was evaluated using 100 fM (green) and 0 fM (gray) IL-6 samples using either PBS or SSC buffers (n=3). Incubating and washing with PBS improved stringency to reduce non-specific binding. The highest signal/background was achieved using 2 ng detection antibody per incubation.

Figure 5.

The quantitative response of dqPCR was evaluated using IL-6 concentrations of 0–5000 fM and a capture time of 2 h (n=3). Digital signals (green squares) exhibited the expected Poissonian response with a LOD of 1.8 fM. Cq values (black diamonds) showed the expected decrease at high concentration when multiple protein molecules were initially present in the microwells.

Figure 6.

The quantitative responses of multiplexed assays were evaluated for (A) let-7a (orange circles) and (B) IL-6 (green squares). The concentration of the target analyte was varied between 0–5000 fM, but the other analyte was present at a constant 500 fM concentration to evaluate potential crosstalk. Concentration-dependent Poissonian responses were observed for each target analyte, while the control analyte exhibited a consistent signal at all concentrations (n=3). These results indicate that crosstalk was not present. LODs for let-7a and IL-6 were 2.5 fM and 3.9 fM, respectively.

Scheme 1.

Cartoon illustrating analyte capture onto beads for multiplexed dqPCR assays. miRNA analysis beads (light red) are pre-functionalized with DNA guides (blue). Guides capture target miRNAs (orange) from the sample and serve as the template in BS-dqPCR. Protein analysis beads are pre-functionalized with encoding dye (red) and capture antibodies (purple). Target protein (dark green) is captured onto beads and then labeled with secondary antibody (light blue) conjugated to a DNA tag (light green) that is amenable for immuno-dqPCR. Beads from both populations are combined for incubation with a biological sample and then stochastically loaded into a microwell array. BS-dqPCR and immuno-dqPCR are conducted simultaneously to measure target miRNA and protein, respectively, using a single detection chemistry. Microwells originally containing a target analyte exhibit high fluorescence after PCR (lime green).