New Method for RNA-Sequencing in Single Cells – Without Killing the Cell
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Researchers present a novel approach to single-cell RNA sequencing (scRNA-seq) called “Live-seq”, which enables the extraction of mRNA from single cells without killing the cell. The method is published in Nature.
An introduction to RNA-sequencing
RNA sequences “mirror” the DNA sequences from which they are transcribed. RNA-sequencing (RNA-seq) – a method that analyzes the total cellular content of RNA (mRNA, rRNA or tRNA, depending on the protocols used) – can therefore help scientists to explore which genes are turned “on” or turned “off” in a cell.
The applications of RNA-seq are vast; it can be used to determine which genes are transcribed in specific states such as disease, or during different developmental stages.
Earlier approaches in RNA-seq adopted a “bulk analysis” approach, whereby multiple cells are extracted from a sample collectively for analysis. The issue here is that the sample could potentially comprise different cell types, making it challenging to conduct cell-specific analyses. The advancement of scRNA-seq has helped to overcome this issue, but requires isolated cells to be lysed – killing them.
A new research study led by Dr. Wanze Chen, postdoctoral fellow at EPFL and Dr. Orane Guillaume Gentil from the Institute of Microbiology at ETZ Zurich, outlines a novel method – “Live-seq” – a minimally-invasive approach to scRNA-seq that keeps cells alive throughout RNA extraction.
How does Live-seq work?
Live-seq works by using a microscopic technique known as fluidic force microscopy, or “FluidFM”. This method utilizes an extremely thin microscopic channel to manipulate small amounts of fluid in a sample underneath a microscope. This way, scientists can insert substances into individual cells, or extract cytoplasm (including mRNA), without the need to harvest them.
“To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations,” the authors wrote in the publication. The method enables researchers to connect a cell’s transcriptome to its molecular phenotype at a given time, which the scientists are referring to as a “temporal transcriptomic analysis”.
“With Live-seq, we can now uniquely address highly interesting and biomedically relevant questions, such as why certain cells differentiate and sister cells do not, or why certain cells are resistant to a cancer drug, while their sister cells are again not,” says Dr. Bart Deplancke, professor and leader of the Deplancke Group at EPFL, and co-author of the study.
Chen and colleagues successfully tested their novel method in macrophages before and after these cells were “activated”, and in adipose stromal cells (a form of stem cell) before they differentiated into fat cells.
Concluding their publication, the researchers state that they anticipate Live-seq will “transform single-cell transcriptomics” and potentially impact other omics technologies, such as single-cell proteomics and metabolomics.
Reference: Chen W, Guillaume-Gentil O, Rainer PY, et al. Live-seq enables temporal transcriptomic recording of single cells. Nature. 2022. doi: 10.1038/s41586-022-05046-9.
This article is a rework of a press release issued by EPFL. Material has been edited for length and content.