Ultrasensitive micro RNA quantification

A system for quantifying nucleic acid at a single molecule level in a biological sample, the system comprising:

• (1) a sub-system for extending the nucleic acid to form an amplicon,
• (2) a subsystem for labeling the amplicon with more than one fluorescent dye to form a labeled nucleic acid amplicon, wherein the labeled nucleic acid amplicon is labeled by:
• (i) hybridization of the nucleic acid amplicon with a fluorescent dye conjugated to a single-stranded nucleic acid or nucleic acid analog,
• (ii) binding or intercalation of single-stranded nucleic acid-specific or double-stranded nucleic acid-specific fluorescent dyes with the amplicon, and/or
• (iii) addition of the fluorescent dye into the nucleic acid amplicon by enzymatic incorporation of a fluorescent dye labeled nucleoside triphosphate or deoxyribonucleoside 5′-triphosphate,
• (3) a fluorescence detection sub-system comprising an excitation source and fluorescence detector for excitation of the fluorescently labeled amplicon and measuring fluorescence emission from individual molecules of the fluorescently labeled amplicon, wherein the excitation source comprises a laser source, an incandescent light source, or an light emitting light source, and
• (4) software to record and analyze the fluorescence emission from the detection subsystem wherein the software to record and analyze the fluorescence emission facilitates minimizing or eliminating aberrant counts from the fluorescence emission of the fluorescently labeled single-stranded DNA amplicon by correlating signals from multiple fluorescent labels with optical scattering events using multiparametric gating.