Point-of-care diagnostic tools to detect circulating microRNAS as biomarkers of disease

Abstract

MicroRNAs or miRNAs are a form of small non-coding RNAs (ncRNAs) of 19-22 nucleotides in length in their mature form. miRNAs are transcribed in the nucleus of all cells from large precursors, many of which have several kilobases in length. Originally identified as intracellular modulators of protein synthesis via posttranscriptional gene silencing, more recently it has been found that miRNAs can travel in extracellular human fluids inside specialized vesicles known as exosomes. We will be referring to this miRNAs as circulating microRNAs. More interestingly, the miRNA content inside exosomes changes during pathological events. In the present review we analyze the literature about circulating miRNAs and their possible use as biomarkers. Furthermore, we explore their future in point-of-care (POC) diagnostics and provide an example of a portable POC apparatus useful in the detection of circulating miRNAs.

Figure 1.

Sensitivity of the total internal reflection fluorescence (TIRF)-based microarray platform. (a) Examples of the signal detected by a charge-coupled device (CCD) camera arising from microarray spots developed to detect synthetic DNA sequences (for details please refer to the original publication [42]). To verify the reproducibility of the fluorescent signal the MB was spotted in tandems of 10 (left to right spots). Increasing concentrations of the target sequence were introduced in the system (from 0 used as control, 0.1, 1, 10 and 100 nM). The red circle indicates the region of interest (ROI) from which fluorescence was measured over time; (b) Mean fluorescence values obtained from the ROIs on the 10 fluorescent spots in the microarray illustrated in (a). The plots illustrate the time courses of association of the sample to the molecular beacons (MB). Dotted red line shows the background level. Each of the concentrations of the target tested is indicated at the right of the panel; and (c) fluorescence intensity (in arbitrary units, AU) illustrating the mean ± standard deviation obtained from the 10 fluorescent spots in the microarray shown in (a). Values were measured at the time indicated by the gray rectangle in (b). Notice that the limit of detection was around 0.1 nM. Figure adapted from [42], with permission from the authors.

Figure 2.

Portable PCR-microarray combined system with potential use in POC diagnostics (a) drawing illustrating the dual chamber system, left a needle is used to apply the sample which enters into the PCR (first) chamber. A heating jacket keeps the temperature at 60 degrees Celsius, the temperature at which the Bst DNA polymerase has its highest activity. The PCR chamber contains already a master mix with primers, oligonucleotides and PCR reaction buffers. Sample is maintained in the PCR chamber for 60 min, after which it is transferred into the microarray chamber (right). The microarray chamber contains the coverslip with low-density molecular beacons (MB) printed using a low melting point agarose solution (MB shown in yellow). (b) Illustration of the array of avalanche photodiodes arranged to detect fluorescence emitted from the MB in the microarray spots. Photodiodes are connected to an integrated circuit, which reads the current generated by the photons bouncing on the photodiode. The current is then send to a liquid crystal display (LCD) screen for real-time viewing of data and stored on a microSD card for later analysis and plotting. (c) Examples of curves illustrating the time courses of relative signal obtained under the different experimental conditions. As positive control an oligonucleotide that matched perfectly to the sequence of a control MB was utilized. For simplicity sake, only the time courses of detection of two miRNAs are depicted (miR-21 in green and miR-103 in red). The blue line illustrates the values obtained applying the same sample but skipping the PCR reaction step (miR-21 without PCR in blue). Notice that signal values are within the noise value. Negative control is a MB for which no matching sequence was present. This value reflects the autofluroescence of the MB alone (without the target present). (d) Plots illustrating the mean and standard deviation values obtained from 5 independent reactions for the 6 miRNAs explored in this study (miR-21, miR-25, miR-103, Let7a, miR-34 and miR-206). The dotted line represents the level of autofluroescence (background) signal. Notice that without the PCR amplification step, miR-21 cannot be detected (blue bar). The same sample subjected to the PCR amplification step generates a robust signal for this miRNA (green bar).

Figure 2.

Portable PCR-microarray combined system with potential use in POC diagnostics (a) drawing illustrating the dual chamber system, left a needle is used to apply the sample which enters into the PCR (first) chamber. A heating jacket keeps the temperature at 60 degrees Celsius, the temperature at which the Bst DNA polymerase has its highest activity. The PCR chamber contains already a master mix with primers, oligonucleotides and PCR reaction buffers. Sample is maintained in the PCR chamber for 60 min, after which it is transferred into the microarray chamber (right). The microarray chamber contains the coverslip with low-density molecular beacons (MB) printed using a low melting point agarose solution (MB shown in yellow). (b) Illustration of the array of avalanche photodiodes arranged to detect fluorescence emitted from the MB in the microarray spots. Photodiodes are connected to an integrated circuit, which reads the current generated by the photons bouncing on the photodiode. The current is then send to a liquid crystal display (LCD) screen for real-time viewing of data and stored on a microSD card for later analysis and plotting. (c) Examples of curves illustrating the time courses of relative signal obtained under the different experimental conditions. As positive control an oligonucleotide that matched perfectly to the sequence of a control MB was utilized. For simplicity sake, only the time courses of detection of two miRNAs are depicted (miR-21 in green and miR-103 in red). The blue line illustrates the values obtained applying the same sample but skipping the PCR reaction step (miR-21 without PCR in blue). Notice that signal values are within the noise value. Negative control is a MB for which no matching sequence was present. This value reflects the autofluroescence of the MB alone (without the target present). (d) Plots illustrating the mean and standard deviation values obtained from 5 independent reactions for the 6 miRNAs explored in this study (miR-21, miR-25, miR-103, Let7a, miR-34 and miR-206). The dotted line represents the level of autofluroescence (background) signal. Notice that without the PCR amplification step, miR-21 cannot be detected (blue bar). The same sample subjected to the PCR amplification step generates a robust signal for this miRNA (green bar).

Figure 3.

Identification of miRNAs having one or two mismatches with our TIRF-based microarray platform (a) mature sequences from Let7a1 (black), Let7b (red) and Let7c (green). Gray rectangles show the nucleotide mismatches found between Let7a1 (used as reference) and Let7b and Let7c; and (b) Mean values obtained from the readings of 10 spots (10 independent avalanche photodiodes) for each miRNA tested. The vertical arrow points to the time in which the synthetic miRNA was added to the system (either Let7a1, Let7b or Let7c). All miRNAs were added at a final concentration of 1 nM. Colors of the curves correspond to the sequences in (a). The MB printed on the microarray spots corresponds to the complementary sequence of Let7a1 (perfect matching).