The genome, epigenome, transcriptome and proteome are usually distinct for each of the 40 trillion cells in the human body. This cellular heterogeneity means that to fully understand physiology, and the etiology of thousands of genetic-origin diseases afflicting mankind, biologists must have an understanding of the individual cellular origin of disease. If an experiment is to yield usable results, cells must be isolated (from other cell types) and purified and debris removed, all while not altering the cell state and with maximal recovery rates (yields). Here at LevitasBio, we are dedicated to optimizing those critical, initial stages of cell isolation and purification, that is the full scope of sample preparation, steps antecedent to the most advanced sequencing and computational techniques used in laboratories around the world.
Most cells (excepting red blood and cornified cells) have a nucleus which contains the genomic DNA as well as the mRNA, ribosomes, and transfer RNA (tRNA) needed for gene expression. The nucleus is surrounded by a double membrane, or nuclear envelope, that separates its contents from the rest of the cell. The mRNA and assembled ribosomes (complexes of RNA and protein) are exported out of the nucleus, through pores in the nuclear membrane, to the surrounding cytoplasm where translation occurs.
Isolation and purification of cell nuclei is essential in laboratory studies of when and how proteins and other molecules are distributed between the nucleus and the cytoplasm. For example, isolated nuclei are used to identify protein binding sites on DNA through chromatin immunoprecipitation (ChIP) or DNAse I accessibility assays, and to study chromatin structure via ATAC (assay for transposase-accessible chromatin) sequencing. Separating the nucleus from the rest of the cell is also important to the study of nuclear proteins such as telomerase or histones, and to the study of the fractionation of proteins between the nucleus and the cytoplasm, via immunoblotting.
Increasingly, nuclei are isolated and purified for single-cell gene expression studies. As noted, single-cell studies are essential to accurately measure the heterogeneity of a cell population, such as the variety and distribution of cell types present in a sample of peripheral blood. Single-cell studies can reveal the heterogeneity present in a tumor or tissue sample such that differential responses to drugs or stimuli can be pinpointed. However, it can be difficult or impossible to obtain single cells from some sample types, and in these scenarios, scientists may choose to use isolated nuclei to study the heterogeneity of their sample instead.
The isolation and purification of cell nuclei must be optimized for every tissue and cell type. For solid tissues, the tissue must first be homogenized either chemically or physically. Then, the cell membrane must be lysed to release the nuclei. Microscopic examination is usually used to ensure that cells are completely lysed but that the nuclei remain intact. Finally, the nuclei must be separated from the resulting cellular debris. The optimal way to remove debris will depend on the tissue and cell type, and on the experimental design: in some cases only the nuclei will be needed but in other cases different organelles will be collected as well to compare the subcellular localization of a component between the nucleus and cytoplasm. Purification approaches include various types of filtration often accompanied by differential centrifugation. Frequently, nuclei stick together and aggregate so for single-nuclei studies, buffers must be optimized to prevent aggregation and purification protocols must remove the aggregates.
We are continually evolving the LeviCell™ system to perfect laboratory techniques and protocols that reduce the cost and improve the quality of sample preparation. This blog series will highlight where and how the LeviCell system can optimize nuclei preparation workflows. Next up: “Working with Single Nuclei – Research Notes from the Field: A weekly account of noteworthy laboratory experiments, and research goals, collected from labs around the world”.