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Single-Cell Spatiotemporal Method Enables Serial mRNA Extraction, RNA-seq From Same Cell


NEW YORK – Researchers at the University of California, Santa Cruz have pioneered a method for repeatedly sampling messenger RNA (mRNA) from single cells without killing the cells in question.

The method, which was recently published in the journal PLoS One, relies on automated nanopipetting from slide-mounted, intact tissue and mRNA capture using a just-launched magnetic mRNA purification and sequencing library preparation platform from nanoparticle assay developer Nvigen.

"[The] nanopipette technology that we have developed is capable of analyzing a single cell while [that] cell is alive," Nader Pourmand, professor of biomedical engineering at UCSC and the study's senior author said via email. "We have shown that we can aspirate, inject, and sense target molecules including nucleic acid, protein, and small molecules such as glucose, [reactive oxygen species], and pH."

The platform is made fully automated and capable of high-throughput sequencing by integrating the nanopipettes with the SU10 controller unit from Yokogawa Electric.

In the proof-of-concept study, Pourmand's group aspirated 22 cells within a breast cancer tissue slice and selectively captured the mRNA using Nvigen's proprietary antibody-coated magnetic nanoparticles. The resulting RNA-seq data showed high alignment rates of approximately 80 percent with two control tissue samples. 

Hundreds of genes were sequenced from each cell at each aspiration, and the genes sequenced showed high correlations from one sampling to the next, indicating that the small amount of mRNA withdrawn from each cell did significantly affect gene coverage.

Pourmand's lab had established the underlying method of sampling material from single cells while maintaining cell viability in 2014 and the more recent study represents a way to reliably do so at scale. Accomplishing this required surmounting certain technological challenges involved with fluid mechanics and the handling of intact tissue.

"Since we were using a tissue slice, there is a higher resistance to current flow that arises from the thickness of the slice that consists of both the tissue and fixing agent and substrate. Aspiration of the intracellular components using a nanopipette requires precise application of the required potential," said Aihua Fu, CEO of Nvigen, which had supported this study with funds from a National Institutes of Health Small Business Innovation Research grant and through the use of its recently launched Nanoparticle Enhanced Spatial and Temporal (NERNST)-Seq mRNA capture and sequencing library preparation kit.

Fu also commented that Nvigen developed the protocol for making tissue samples compatible with Pourmand's nanopipetting platform.

To achieve the necessary precise control in a high-resistance environment, the collaborators used a third electrode that acted as a feedback mechanism to help maintain and control the potential across the nanopipette.

Where most scRNA-seq methods involve killing the cell from which messenger RNA is taken, the UCSC method leaves cells alive for further sampling, taking only between 1 and 5 percent of the cell's mRNA per sampling event.

While the length of time cells remain viable is heavily influenced by lab- and experiment-dependent factors, Pourmand commented that there are limits on how frequently sampling can occur.

"If we sample the cells every 15 minutes, it does not change cell either morphology or activity," he said. "If [intervals are] less than 15 minutes then cells start changing morphology. [Taking] out more than 1 percent of cell content also affects cells."

The utility of longitudinal data in single-cell spatial transcriptomics is well known and numerous methods attempt to address it. As Pourmand suggests in his group's published study, theirs does appear to be the only technique currently available that enables repeat sampling of the same cell at scale.

Transcriptome in vivo analysis (TIVA), for example, enters cell membranes via a cell-penetrating peptide. Upon activation, a light-activated biotin tag is used to bind the poly-A tails of mRNA, capturing mRNA within live cells and intact tissues in a relatively noninvasive manner. While this technique allows scientists to explore single-cell transcriptomes in the context of their natural microenvironment and with clear spatial resolution, it is relatively low throughput with regard to the number of cells that can be analyzed.

Another method called Geo-seq combines scRNA-seq with laser capture microdissection. In this way, cells of interest are geographically marked by imaging then unwanted cells are cut away via infrared or ultraviolet capture systems. The mRNA from target cells is then harvested and sequenced. As with TIVA, however, this is not an easily scalable technique. It also risks contamination from neighboring cells and killing the cells of interest during mRNA collection.

Nvigen's Fu said the company is exploring potential commercial opportunities with Pourmand's lab but that it is too early to comment on them at this time.

The Santa Clara, California-based company has applied its magnetic field gradient-controlled nanoparticle expertise to cell-free DNA extraction. In 2021, the company presented data demonstrating that its Nvigen X Cancer Precision Profiling assay accurately monitors dynamic changes in circulating tumor DNA.

In the current proof-of-concept study, NERNST-Seq was compared to other library preparation methods and resulted in significantly higher sequencing library yields compared to other methods.

Fu sees NERNST-Seq applications in therapeutic response monitoring, drug selection for precision oncology, and tumor evolution studies, among others. The company has begun marketing NERNST-Seq but, with the paper so recently published, has not yet seen it put to use by a customer. Fu commented that Nvigen is currently discussing several potential collaborations with oncologists.

"We are applying for more grants to run more application studies that will help address specific biomedical questions such as efficient drug screening, drug resistance mechanism, cancer metastatic profiling, [and] cancer-immune cell interactions," Fu said.

Pourmand, meanwhile, is exploring the multiomic potential of the integrated nanopipetting and NERNST-Seq method.

"Efforts are underway to be able to detect multiple different targets at once in a single cell," he said.