Simultaneous detection of target protein and target nucleic acids in a single cell
View Patent ↗Methods, reagents, and kits for detection and analysis of nucleic acids are provided. The methods can be used in conjunction with a proximity extension assay for protein detection to provide a multiplex assay to detect both nucleic acids (e.g., RNA) and proteins.
1. A kit for performing a multiplexed assay of RNA and protein from a sample, the kit comprising:
a) at least one pair of protein-detecting proximity probes, wherein each pair of protein-detecting proximity probes comprises:
a first protein-detecting probe comprising a first antibody that binds to a target protein joined to a first polynucleotide that comprises an I segment at the 3′ end that is complementary to an I segment on the 3′ end of a second probe; and
a second protein-detecting probe comprising a second antibody that binds to the target protein joined to a second polynucleotide that comprises an I segment complementary to the I segment at the 3′ end of the first polynucleotide of the first probe;
wherein binding of the first antibody to the target protein and binding of the second antibody to the target protein allows the I segment of the first protein proximity probe to hybridize to the I segment of the second protein proximity probe to form a duplex;
b) a polymerase for extending the duplex to provide an extended product; and
c) primers that together amplify the extended product, or subregion thereof; and amplify two or more target nucleic acid sequences in the same reaction mixture.
2. The kit of claim 1 , wherein the primers are packaged as an admixture in the same composition.
3. The kit of claim 1 , further comprising one or more labels for detecting amplicons produced in step c).
4. The kit of claim 1 , further comprising reagents to isolate single cells.
5. The kit of claim 1 , further comprising reagents to lyse single cells.
6. The kit of claim 1 , further comprising a microfluidic device for isolating and lysing single cells.
7. The kit of claim 1 , wherein the kit comprises compatible probes provided in admixture.
8. The kit of claim 1 , further comprising reagents for performing a quantitative amplification reaction.
9. The kit of claim 8 , wherein the quantitative amplification reaction is qPCR.
10. The kit of claim 1 , wherein the polymerase is a DNA polymerase having 3′ exonuclease activity.
11. The kit of claim 1 , further comprising a DNA polymerase for performing step (c) that is different from the DNA polymerase employed in the extension reaction of step (b).
12. The kit of claim 11 , wherein the DNA polymerase for performing step (c) is a thermostable polymerase.
13. The kit of claim 1 , further comprising reagents for detecting a target RNA sequence and an amplicon obtained by amplifying an extended product of a nucleic acid proximity extension reaction.
14. The kit of claim 1 , wherein RNA of a cell in the sample comprises the target nucleic acid sequences.
15. A method of evaluating a single cell for RNA and protein of interest, the method comprising performing a multiplexed assay on a single cell to detect RNA and protein with a kit comprising:
a) at least one pair of protein-detecting proximity probes, wherein each pair of protein-detecting proximity probes comprises:
a first protein-detecting probe comprising a first antibody that binds to a target protein joined to a first polynucleotide that comprises an I segment at the 3′ end that is complementary to an I segment on the 3′ end of a second probe; and
a second protein-detecting probe comprising a second antibody that binds to the target protein joined to a second polynucleotide that comprises an I segment complementary to the I segment at the 3′ end of the first polynucleotide of the first probe;
wherein binding of the first antibody to the target protein and binding of the second antibody to the target protein allows the I segment of the first protein proximity probe to hybridize to the I segment of the second protein proximity probe to form a duplex;
b) a polymerase; and
c) primers that together amplify the extended product or subregion thereof and amplify two or more target nucleic acid sequences in the same reaction mixture.