MicroRNAs (miRNAs) are short RNA molecules (18–25 nucleotides) that were recently proven to play an important role in the regulation of cellular processes, and their abnormal expression is associated with pathologies such as cancer. A change in the cellular status is typically associated with a simultaneous change in the level of several miRNAs. For example, abnormal expression of two miRNAs was found to be indicative of colorectal cancer in humans. Therefore, both the study of the biological role of miRNA and the use of miRNA for informative disease diagnostics require accurate quantitative analysis of multiple miRNAs. Most methods of miRNA detection are indirect (e.g. PCR, microarrays, SPR, next generation sequencing, etc.), that is, they require chemical or enzymatic modifications of miRNA prior to the analysis. Not only do these modifications make the analysis more complex and timeconsuming but they also reduce the accuracy of the method owing to different efficiencies for modifications of different miRNAs. There are a few direct methods that do not require any modification of the target miRNA. Northern blotting does not require any modifications, however, the method can be tedious and although it can be quantitative its sensitivity is limited. Signal-amplifying ribozymes, in situ hybridization, bioluminescence detection, and two-probe single-molecule fluorescence are other direct miRNA detection methods however, the first two methods are only semi-quantitative while the latter two can hardly be used for multiple miRNAs. Cheng et al. used rolling-circle amplification (RCA), which does not require modification of the miRNA to detect low concentrations of miRNA and can be run in parallel; however, the process is tedious taking over 8 h and the amplification step can potentially lead to biases in quantitation. Thus, there is currently no method for direct quantitative analysis of multiple miRNAs. Herein we report the first direct quantitative analysis of multiple miRNAs (DQAMmiR). DQAMmiR uses miRNAs directly, without any modification, and accurately determines concentrations of multiple miRNAs without the need for calibration curves. This approach was achieved using a capillary-electrophoresisbased hybridization assay with an ideologically simple combination of two well-known separation-enhancement approaches: 1) drag tags on the DNA probes, and 2) single strand DNA binding protein (SSB) in the buffer. In this proof-of-principle work, we developed DQAMmiR for three miRNAs (mir21, 125b, 145) known to be deregulated in breast cancer. DQAMmiR opens the opportunity for simple, fast, and quantitative fingerprinting of up to several tens of miRNAs in basic research and clinical applications. The availability of suitable commercial instruments for DQAMmiR makes the method practical for a large community of researchers. We based DQAMmiR upon a classical hybridization approach, in which an excess of labeled DNA probes are bound to their complementary miRNA targets. Electrophoresis can be used to efficiently separate oligonucleotides, but simultaneously separating the hybrids from each other and from the unbound probes is challenging and so far has not been achieved. We solved the separation problem through a combination of two well-known mobility-shift approaches: 1) drag tags on the probes and 2) single strand DNA binding (SSB) protein in the buffer. This hypothetical approach is illustrated in Figure 1, in which the miRNAs and their complimentary ssDNA probes are shown as short lines of the same color, drag tags are shown as parachutes, fluorescent labels are shown as small green circles, and SSB is shown as a large black circle. In the hybridization step, an excess of the probes is mixed with the miRNAs, thus leading to all miRNAs being hybridized but with some probes left unbound to miRNA. A short plug of the hybridization mixture is introduced into a capillary prefilled with an SSBcontaining buffer. SSB binds all ssDNA probes but does not bind the double-stranded miRNA–DNA hybrid. When an electric field is applied, all SSB-bound probes move faster than all the hybrids (SSB works as a propellant). Different drag tags make different hybrids move with different velocities. SSB-bound probes, however, can move with similar velocities if the drag tags are small with respect to SSB. In such a case, a fluorescent detector at the end of the capillary generates separate signals for the hybrids and a cumulative signal (one peak or multiple peaks) for the excess of the probes. The amounts of the different miRNAs are finally determined from integrated signals (peak areas in the graph) by a simple mathematical approach. We reserve the term of direct quantitative analysis of multiple miRNAs and its abbreviation of DQAMmiR for the specific approach described above. To experimentally test the viability of our hypothetical DQAMmiR, we decided to use three miRNAs known to be deregulated in breast cancer: mir21 (5’-UAGCUUAUCAGA CUGAUGUUGA-3’), mir125b (5’-UCCCUGAGACCCUAACUU GUGA-3’), and mir145 (5’-GUCCAGUUUUCCCAGGAAUCCC U-3’). Three ssDNA probes were designed and all are labeled with Alexa 488 at the 5 end; the 3 end was reserved for drag tags. To separate the three hybrids we needed only two probes modified with drag [*] D. W. Wegman, Prof. S. N. Krylov Department of Chemistry, York University 4700 Keele Street, Toronto, Ontario M3J 1P3 (Canada) E-mail: skrylov@yorku.ca