Compositions and kits for molecular counting
Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.
1. A method for the absolute quantification of copies of mRNA molecules in a sample, comprising:
(a) stochastically labeling mRNA copies of each of two or more genes of interest in a sample with a plurality of oligonucleotide tags to produce a plurality of labelled-mRNA copies, wherein:
(i) the number of mRNA copies of each of the two or more genes of interest is at least one;
(ii) the plurality of oligonucleotide tags each comprises an oligodT sequence, a universal primer binding site, and an identifier region;
(iii) the plurality of oligonucleotide tags comprises at least 1000 oligonucleotide tags having identifier regions of different sequences for determining the number of mRNA copies of each of the two or more genes of interest;
(iv) a number of oligonucleotide tags having identifier regions of different sequences in the plurality of oligonucleotide tags is at least 5 times greater than the number of mRNA copies of any of the two or more genes of interest; and
(v) wherein stochastically labeling mRNA copies comprises attaching the oligonucleotide tags to the mRNA copies by hybridization;
(b) conducting a first strand synthesis reaction by contacting the plurality of labelled-mRNA copies with a reverse transcriptase enzyme to produce a plurality of single-stranded labelled-cDNA molecules, wherein the first strand synthesis reaction is not performed on a solid surface;
(c) amplifying the plurality of single-stranded labelled-cDNA molecules to produce a plurality of double-stranded labelled-cDNA molecules, wherein the amplifying comprises annealing a first universal primer to the universal primer binding site of the single-stranded labelled-cDNA molecules and annealing a first target-specific primer to the single-stranded labelled-cDNA molecules;
(d) conducting a nested PCR reaction on the plurality of double-stranded labelled-cDNA molecules to produce a plurality of nested PCR labelled-amplicons, wherein conducting the nested PCR reaction comprises annealing a second universal primer to the universal primer binding site of the double-stranded labelled-cDNA molecules and annealing a second target-specific primer to the double-stranded labelled-cDNA molecules, wherein the second target-specific primer anneals downstream of the first target-specific primer; and
(e) detecting at least a portion of amplicons of the nested PCR labelled-amplicons to count the number of different identifier regions associated with each of the labeled mRNA copies, thereby counting the absolute number of mRNA copies of each of the two or more genes of interest in the sample.
2. The method of claim 1 , wherein the sample is from a single cell.
3. The method of claim 1 , wherein the sample is from less than 50 cells.
4. The method of claim 1 , wherein said step (a) is not performed on a bead surface.
5. The method of claim 1 , wherein said detecting comprises determining the sequence of at least a portion of at least one of the nested PCR labelled-amplicons, a complement thereof, a reverse complement thereof, or any combination thereof.
6. The method of claim 1 , wherein said detecting comprises using an array detector, fluorescent reader, non-fluorescent detector, CR reader, sequencer, or scanner.
7. The method of claim 1 , wherein said detecting comprises hybridizing said nested PCR labelled-amplicons to a solid support.
8. The method of claim 7 , further comprising determining the sequence of at least a portion of at least one of said nested PCR labelled-amplicons.
9. The method of claim 1 , wherein the number of oligonucleotide tags having identifier regions of different sequences in the plurality of oligonucleotide tags is at least 10,000.
10. The method of claim 1 , wherein said detecting comprises hybridization chain reaction (HCR).