Spotlight on Laboratory Experiments and Protocols

Last week, we discussed labs’ increasing emphasis on discovering the individual cellular origin of disease. Although both single-cell RNA sequencing (scRNA-Seq) and single-nucleus RNA sequencing (snRNA-Seq) are used to characterize the transcriptional profile of individual cells, scRNA-Seq analyses are confounded in, for example, the case of large cells (>50 µm) or cell adherence. Enzymatic and mechanical methods for dissociation can also alter gene expression. Most single cell isolation for scRNA-Seq requires fresh tissue, which is not always available for human or animal model experiments. There is an increasing focus on nuclei isolation and purification because nuclei can be isolated from cells without regard to size or “freshness” (i.e., frozen tissues can be processed). Purification processes, even those utilizing some mechanical force, do not affect gene expression or, at least, the effects are often minimal enough.

Starting with the below account from researchers at the Leibniz Institute of Resilience Research and the Institute of Human Genetics, University Medical Center of the Johannes Gutenberg University Mainz, we will discuss a newly reported method, experiment, or review that involves isolation and purification of nuclei from mammalian cells and tissues or plant tissues. For many of the cell types discussed, the LeviCell instrument will be appropriate for front end live-dead separation, debris clean-up, and ultimately any degree of cell, or nuclei, purification and enrichment that is required in the experimental design. We hope these regular updates showcasing new scRNA-Seq experiments will further awareness and progress in the industry. If you have a particular research focus, or are wondering if your sample is amenable to purification via our technology, please send us a request and our experts will investigate the problem for you.      

INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison

In “INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison” (Chongtham, M.C.; Butto, T.; Mungikar, K.; Gerber, S.;Winter, J. INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison. Int. J. Mol. Sci. 2021, 22, 5335. https://doi.org/10.3390/ijms22105335) researchers provide a roadmap, and list very specific qualitative and quantitative criteria, that can be used to determine whether Isolation of Nuclei Tagged in Specific Cell Types (INTACT) or Fluorescence-Activated Nuclei Sorting (FANS) will best meet experimental goals. FANS and INTACT have been, to date, used somewhat interchangeably and thus this article is noteworthy as one of a very few papers which provide comparative data on the isolation efficiency and downstream molecular effects of the two techniques.

Researchers compared the physical and molecular attributes of sfGFP+ nuclei isolated by the two methods—INTACT and FANS—from the neocortices of Arc-CreERT2 x CAG-Sun1/sfGFP animals. The study includes a table providing a complete summary, based on 8 criteria, of the qualitative and quantitative differences between INTACT and FANS. In addition, the researchers report the results of a sizable literature review, covering over 300 recent articles, that resulted in a concise comparison of nuclei sorting techniques (published in a supplement to the article).

The study identified differences in “efficiency of sfGFP+ nuclei isolation, nuclear size as well as transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) states.” As a consequence, they recommend use of only one method, within a given study, to eliminate bias in outcomes. As other studies have found although FANS is common to isolate specific cells/nuclei, the often high hydrodynamic pressure could adversely affect the cells/nuclei. The researchers compared INTACT versus FANS insofar as effects on nuclei structure/integrity. They also assessed the transcriptional and chromatin accessibility signatures looking at different library preparation strategies for the nuclear transcriptome. Overall, if the metric is processing speed and costs for multiple samples, INTACT was considered to be more advantageous compared to FANS. However, readers are urged to review Table 2 in the study which, again, provides a very nice overview of the pro and cons of both techniques.

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