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New Study Shows Multiplex Detection of RNA, Protein in Single Cells


NEW YORK (GenomeWeb) – Scientists at Sweden's Uppsala University have described a new method for multiplex detection of both RNA and protein for single-cell biology.

Led by co-first authors Spyros Darmanis and Caroline Gallant and senior author Ulf Landegren, the researchers used proximity assays and pairs of antibodies conjugated to oligonucleotides to detect dozens of targets. They published their proof-of-concept study in Cell Reports in December.

When the antibodies bind to target, the conjugated oligos will be in proximity, making a new DNA reporter that can be amplified, detected, and quantified by qPCR.

"This method of proximity extension allows high multiplexing with very little cross reacting which you would see in a standard ELISA," Gallant told GenomeWeb, allowing up to 96-plex, up from eight-plex. The method uses Fluidigm BioMark real-time PCR, so it can analyze up to 96 targets at once; however, the researchers were able to analyze up to 78 targets at once.

"We could do either a lot more cells or a lot more targets," she said, "We're just limited by readout right now."

The new method could help characterize sub-populations of cells, Gallant said. "With single cells, you want to look at as many different parameters to know what's going on." Even though there are robust methods using RNA to look at cell state and cell identity, "you have no idea what that means when you translate [those RNAs] and look at protein levels," she said. "There's more and more evidence that RNA isn't a perfect representation of protein amount and protein function in cells."

Looking at both RNA and protein might reveal how transcription works in single cells, by correlating how RNA levels change over time with the protein levels. "That's been difficult to do except in situ, where you're limited to four-plex," Gallant said.  Since proteins are the drivers of cell function — and there is more protein in a cell than RNA — it would be useful just to be able to see what their levels are, she said.

While scientists can use cytometry time-of-flight instruments, such as  Fluidigm's Helios platform, to detect proteins, those are limited to about 40-plex, Gallant said. The new method could double that.

To demonstrate the multiplexed capabilities of the proximity assay-based method, the researchers looked at the effect of an investigational therapeutic agent, BMP4, on cell cultures of a type of brain tumor, glioblastoma.

The drug is thought to make some of the cells in glioblastoma populations less proliferative. "We knew that there are cells that wouldn't respond so we're interested in understanding them at a single-cell level," Gallant said. "What contributes to BMP4 resistance? Which are the cells that are not responding?"

Using their method, the researchers found a sub-cluster of cells that responded differently to BMP4, Gallant said, remaining proliferative when exposed to it and expressing a number of markers that weren't know to be related to the drug. Moreover, the protein analysis proved to be essential to detecting that sub-cluster.

It was a simple proof of concept in just one cell line, Gallant said, but suggested that the method could work for lots of targets, as long as there existed antibodies amenable to conjugation. For the RNA targets, she said any targeted gene expression assay would work, though they used off-the-shelf TaqMan assays.

Senior author Ulf Landegren is a co-founder of O-Link Bioscience, which has already commercialized the proximity assays.