METHODS AND SYSTEMS FOR SINGLE-MOLECULE RNA EXPRESSION PROFILING

摘要

In general, the invention features methods and systems for expression profiling of RNA from a sample, e.g., a single cell, based on the simultaneous measurement of continuous incorporation of fluorogenic nucleotides into complementary nucleic acids. Methods for profiling RNA (e.g., of prokaryotic, eukaryotic, or viral origin) expression in a sample may include disposing, in each of a plurality of optionally sealed microreactors, a mixture in solution phase comprising a single copy of a target RNA (or DNA copy thereof) from a sample (e.g., a single cell), a nucleic acid replicating catalyst, and a mixture of nucleotides having a first nucleotide with a first label that is substantially non-fluorescent until after incorporation into a nucleic acid based on complementarity to the target; allowing continuous template-dependent replication of the targets in the microreactors; sequencing the targets in the microreactors by detecting in real time the individual incorporation of the first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from the first label; and determining from the sequencing the level of expression of RNA in the sample.