DNA barcodes enable high throughput RNA and protein detection in deep tissue

11 Mar 2025, 16:44 by Howard Hughes Medical Institute

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Credit: Gandin and Kim et al.

For the Liu Lab, necessity is truly the mother of invention. The researchers were examining how the 3D organization of the genome controls development and needed to image hundreds of RNA molecules in a thick tissue sample to understand where and how genes were being expressed in cells. There was just one problem: There weren't any tools that were up to the task.

One technique could image lots of RNA molecules, but only in a thin layer of cells. Another method could image in deep tissue but could only detect three or four molecules in a single sample.

So, the team at HHMI's Janelia Research Campus decided to build their own tool. The result is an innovative new technique that uses a novel DNA barcode system to track hundreds of RNA and protein molecules in single cells within thick biological samples, providing researchers with a full picture of how these structures are organized inside tissues. The research is published in the journal Science.

RNA molecules carry instructions from DNA for making proteins that carry out much of the work of living cells. Knowing where these molecules are located in complex tissues is a critical part of understanding where and how genes are being expressed across different regions and cell types. This information enables researchers to decipher how genes function in different parts of an organism, how they enable development, and how they might be altered in diseases.

Beyond its use in biology and neuroscience, the new method could also potentially be used in diagnostic imaging, according to the researchers.

"I think it will be a gamechanger very broadly, not just for people in my field," says Janelia Group Leader James Liu. "It was a tool developed to answer a very obscure question, but I think all biologists can use the technique in their favorite samples."

Janelia researchers developed an innovative new technique—cycleHCR—that uses a novel DNA barcode system to track hundreds of RNA and protein molecules in single cells within thick biological samples, providing researchers with a full picture of how these structures are organized inside tissues. This movie shows cycleHCR imaging of eight protein targets in a hippocampal slice, as part of the joint RNA and protein imaging. Credit: Gandin and Kim et al.

Depth and throughput

The new imaging tool, called cycleHCR, builds on a previously developed technique called Hybridization Chain Reaction, or HCR. The technique assembles multiple fluorophores on a target that shine like a bright beacon when imaged by a fluorescence microscope, enabling researchers to see molecules in single cells deep inside tissues.

But HCR is limited by current fluorophores, which, because of the wide spectrum they cover, only allow three or four colors to be used at one time. This means researchers can only detect a handful of molecules in a sample, making it difficult to get a full picture of how they are organized within tissues.

To overcome this, the team designed novel DNA barcodes that they could attach to the targets. Just like barcodes on products in a supermarket, which designate every individual type of item in the store, the unique DNA barcodes allow the researchers to tag each type of molecule in the sample.

Each barcode contains two parts. When the two pieces match, the target is amplified by the HCR technique. The two parts of the barcode ensure the tags are specific enough to detect individual types of RNA molecules.

The barcodes were also designed to be easily removed, so multiple rounds of HCR can be performed on the same sample. The initial round of imaging uses three barcodes, picking up three RNA molecules in three different colors. These barcodes are removed, and a second round of imaging uses three different barcodes and so on for multiple rounds, eventually allowing the discovery of an unlimited number of targets in a single sample.

Automation of cycleHCR procedures. Credit: Science (2025). DOI: 10.1126/science.adq2084

"We modified the split amplification chain reaction technique in a way that now we are adding barcoding to it where we can detect hundreds, potentially even thousands of RNAs, with these multi rounds," says Valentina Gandin, a Senior Scientist in the Liu Lab who co-led the research. "The barcoding was a novelty that we added to this."

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In addition to detecting RNA, the researchers developed a way to use the same barcodes to detect proteins, allowing researchers to better understand how both RNA and proteins are organized in tissues.

The team also automated the system, enabling the researchers to detect up to a dozen molecular species in a single day without having to constantly monitor the process. Additionally, Postdoctoral Scientist Jun Kim, a co-first author of the new study, developed analysis methods that map where genes are expressed spatially and help researchers make sense of the raw data. The Liu Lab also worked with the Scientific Computing Software support team at Janelia and developed an automated pipeline for processing the large amount of imaging data generated.

The researchers worked with other labs at Janelia to use the new method to quantify gene expression in mouse embryos, quantifying 254 genes in a single sample. This enabled the researchers to characterize all the cell types in the embryo and discover new cell types that had not been previously characterized, information that is important for understanding development.

The new technique has already generated attention in the scientific community, and the team is working to enable more labs to use the new tool. They shared all of the barcode sequences so other labs can design their own probes, even if they don't have an automated system. The team is also building a more streamlined version of their prototype system and plan to share their automated platform with the scientific community.

"Eventually we want everybody to use it," Liu says. "We would really like our technique to be broadly spread to enable every scientist to be able to use it."

More information: Valentina Gandin et al, Deep-tissue transcriptomics and subcellular imaging at high spatial resolution, Science (2025). DOI: 10.1126/science.adq2084

Journal information: Science

Provided by Howard Hughes Medical Institute