Ultrasensitive micro RNA quantification

1. 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.

2. The system according to claim 1 wherein extending the nucleic acid is facilitated by enzymatic amplification of the nucleic acid by rolling circle amplification.

3. The system according to claim 1 wherein the excitation source comprises a laser.

4. The system according to claim 1 wherein the fluorescence detection sub-system comprises a microfluidic device with multiple optical sensors to measure fluorescence.

5. The system according to claim 1 wherein the fluorescence detection sub-system comprises a flow cytometer.

6. The system according to claim 1 wherein the amplicon comprises double-stranded DNA, single-stranded DNA, single-stranded RNA or a concatemer of RNA and DNA.

7. The system according to claim 1 wherein the fluorescent dye-conjugated single-stranded nucleic acid comprises primarily single-stranded DNA, single-stranded RNA, single-stranded peptide nucleic acid (PNA) or single-stranded locked nucleic acid (LNA).

8. The system according to claim 1 wherein the fluorescent dye-conjugated single-stranded nucleic acid comprises as a fluorescent dye, one or more light emitting coumarin, cyanine, fluorescein, rhodamine, oxazine, Alexa, ATTO, or BODIPY, fluorescent phycobiliprotein, green fluorescent protein, DsRed dye, light-emitting semiconductor nanocrystal, or light-emitting organic polymer, nanoparticle, or bead.

9. The system according to claim 1 wherein the target nucleic acid for quantitation is double-stranded DNA, single-stranded DNA, micro RNA (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), interfering RNA (siRNA), antisense RNA (aRNA), transfer messenger RNA (tmRNA), tRNA-derived small RNA (tsRNA), rDNA-derived small RNA (srRNA), ribozyme, viral RNA or double-stranded RNA.

10. The system according to claim 9 further comprising a categorizing sub-system for spectrally barcoding the fluorescently labeled single-stranded DNA amplicon to multiplex numerous miRNA sequences.

11. The system according to claim 10 wherein the spectral barcoding is performed using designed template sequences, which result in the generation of complementary amplicons sequences that can be fluorescently labeled in a sequence specific manner.

12. The system according to claim 10 wherein fluorescence labeling results from sequence specific incorporation of fluorescent dye labeled nucleoside triphosphate or deoxyribonucleoside 5′-triphosphate.

13. The system according to claim 10 wherein the spectral barcoding is accomplished using multiparametric single-molecule analysis.

14. The system according to claim 10 wherein the spectral barcodes are differentiated using, at least in part, data from colorimetric and/or ratiometric selections.

15. The system according to claim 1 wherein the more than one fluorescent dye comprises one or more intercalating fluorescent dyes that exhibit a fluorescence enhancement of at least 10 times upon binding of the intercalating fluorescent dye to double-stranded DNA.

16. A system according to claim 15 wherein the one or more intercalating fluorescent dyes comprises one or more cyanine dyes.