US 2016/0108458 A1Published application

Multiplexed detection and quantification of nucleic acids in single-cells

AssigneeThe Board of Trustees of the Leland Stanford Junior University

InventorsAndreas Philipp Frei, Garry P. Nolan, Pier Federico Gherardini, Felice Alessio Bava

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Abstract

Proximity Ligation Assay for RNA (PLAYR) provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. An advantage of PLAYR includes the ability to simultaneously analyze multiple nucleic acids and proteins in single cells, as the method is compatible with conventional antibody staining for proteins, intracellular phosphorylation sites, and other cellular antigens. This enables the simultaneous detection of multiple nucleic acid molecules in combination with additional cellular parameters.

Claims20

Dependent claims

A method for determining the abundance of a target nucleic acid in a single cell, the method comprising:

contacting a fixed and permeabilized cell with at least one pair of oligonucleotide primers under conditions permissive for specific hybridization, wherein each oligonucleotide in the pair comprises:
(i) a target binding region that hybridizes to the target nucleic acid;
(ii) a spacer region that does not bind to the target nucleic acid or to any region of a padlock probe;
and
(iii) a PLAYR1 or PLAYR2 region that specifically binds to a padlock probe;
washing the cell free of unbound primer
contacting the cell with a padlock probe under conditions permissive for specific hybridization, wherein the padlock probe comprises separate polynucleotides of (i) a backbone and (ii) an insert;
contacting the cell with ligase wherein bound backbone and insert polynucleotides are ligated to generate a closed circle;
performing rolling circle amplification using the closed circle as a template and PLAYR1 or PLAYR2 as a primer for a polymerase;
contacting the cell with a detection probe under conditions permissive for specific hybridization;
and
detecting the level of bound detection probes to determine the abundance of the target nucleic acid.

9 more elements

Dependencies are inferred from the claim text.