Simultaneous detection of target protein and target nucleic acids in a single cell
View Patent ↗Methods and reagents for detection and analysis of nucleic acids are provided. The methods employ proximity extension assays for detection of a target nucleic acids of interest, e.g., a target RNA. The method can additionally be used in multiplex assays with a protein proximity extension assay to detect protein.
1. A method of detecting an RNA and a protein in a sample, the method comprising:
(a) incubating a sample in a reaction mixture with a pair of protein-detecting proximity probes comprising a first and a second protein detecting proximity probe where:
the first protein-detecting probe comprises a first antibody that binds to the 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 the second probe; and
the second protein-detecting probe comprises 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;
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) extending the duplex using a DNA polymerase to provide an extended product
(c) amplifying the extended product, or a subregion thereof, using a set of primers that amplify the extended product or subregion thereof; and
(d) detecting an RNA of interest in the sample and the amount of amplicon from amplification of the extended product or subregion thereof.
2. The method of claim 1 , wherein the sample is a single cell.
3. The method of claim 1 , wherein the detecting step comprises a quantitative amplification reaction.
4. The method of claim 3 , wherein the quantitative amplification reaction is qPCR.
5. The method of claim 1 , wherein the DNA polymerase has 3′ exonuclease activity.
6. The method of claim 1 , wherein the amplification of step (c) is performed with a DNA polymerase different from the DNA polymerase employed in the extension reaction of step (b).
7. The method of claim 6 , wherein the DNA polymerase in amplification reaction is a thermostable polymerase.
8. The method of claim 1 , wherein the detecting step comprises a quantitative amplification reaction.
9. The method of claim 8 , wherein the quantitative amplification reaction is qPCR.
10. The method of claim 1 , wherein the detecting step comprises quantifying the amount of amplicon.
11. The method of claim 1 , wherein detecting the RNA of interest in the sample comprises detecting an amplicon obtained by amplifying an extended product of a nucleic acid proximity extension reaction.