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 a gene 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 the gene 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 100 oligonucleotide tags having identifier regions of different sequences for determining the number of mRNA copies of the gene of interest; and
(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;
(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;
(c) amplifying the plurality of single-stranded labelled-cDNA molecules to produce a plurality of double-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; 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 the labeled mRNA copies, thereby counting the absolute number of mRNA copies of the gene of interest in the sample.
2. The method of claim 1 , wherein amplifying the plurality of single-stranded labelled-cDNA molecules to produce a plurality of double-stranded labelled-cDNA molecules 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.
3. The method of claim 1 , wherein conducting a nested PCR reaction on the plurality of double-stranded labelled-cDNA molecules to produce a plurality of nested PCR labelled-amplicons 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.
4. The method of claim 1 , wherein the sample is from a single cell.
5. The method of claim 4 , wherein the single cell is a cancerous cell, a diseased cell, a healthy cell, or an infected cell.
6. The method of claim 4 , wherein the single cell is a cell from a subject suffering from a disease or a condition.
7. The method of claim 4 , wherein the sample is cell lysate.
8. The method of claim 1 , wherein the sample is from less than 50 cells.
9. The method of claim 1 , wherein said step (a) is not performed on a bead surface.
10. 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.
11. The method of claim 1 , wherein said detecting comprises using an array detector, fluorescent reader, non-fluorescent detector, CR reader, sequencer, or scanner.
12. The method of claim 1 , wherein said detecting comprises hybridizing said nested PCR labelled-amplicons to a solid support.
13. The method of claim 12 , further comprising determining the sequence of at least a portion of at least one of said nested PCR labelled-amplicons.
14. 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 1000.
15. 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.
16. The method of claim 1 , wherein said detecting comprises hybridization chain reaction (HCR).
17. The method of claim 1 , wherein the first strand synthesis reaction of (b) is not performed on a solid surface.
18. The method of claim 1 , wherein the first strand synthesis reaction of (b) is not performed on a bead surface.
19. The method of claim 1 , wherein the identifier region is at least four nucleotides in length.
20. The method of claim 1 , wherein the oligonucleotide tag is at least ten nucleotides in length.