Abstract
We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1045-6) contains supplementary material, which is available to authorized users.
Highlights
There has been an explosion of papers that utilize highly-multiplexed single-cell Reverse transcription; specific (RNA) target amplification (STA): Specific (RNA) profiling (through quantitative reverse transcription-polymerase chain reaction [1, 2] or sequencing [3,4,5,6,7,8,9]) to investigate the extent, causes, and consequences of cellular heterogeneity
We identified the possibility of coupling reverse transcription (RT) and proximity extension assays (PEA) oligonucleotide extension into a single step by either reverse transcribing RNA with DNA polymerase or extending the hybridized DNA oligonucleotides in PEA with reverse transcriptase
A DNA polymerization reaction is performed using reverse transcriptase to simultaneously extend the hybridized, complementary oligonucleotides conjugated to the PEA probes and reverse transcribe cellular RNA into complementary DNA (cDNA) using random primers
Summary
There has been an explosion of papers that utilize highly-multiplexed single-cell RNA profiling (through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) [1, 2] or sequencing [3,4,5,6,7,8,9]) to investigate the extent, causes, and consequences of cellular heterogeneity. Given the limited number of RNAs and proteins that can be simultaneously assayed in situ and the noise associated with any one measurement [3, 10], the state-of-the-art has been to quantitatively record the levels of select cell surface proteins (index sort) during the fluorescence-activated cell sorting (FACS)-based isolation of single cells that normally precedes single-cell RNA profiling This and related approaches can effectively link precision single-cell protein measurements – and much of the scientific community’s accrued data and knowledge – to high-dimensional single-cell RNA profiles, enabling deeper insights [1, 10, 21,22,23,24]. These techniques are fundamentally limited in both the number (ntotal ~15 due to spectral overlap [10, 25]) and type of protein targets (extracellular, since the fixation and permeabilization required for intracellular staining can degrade cellular RNA [26, 27]) they can assay
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