Isolation of high quality mRNA from a discrete cell cycle population identified using a nonvital dye and fluorescence activated sorting

Abstract

Flow cytometry (FCM) is useful for measuring DNA content as related to cell cycle position. We have extended this technology by developing a method that measures cellular DNA content using propidium iodide after permeabilization with lysolecithin. This technique maintains cell integrity such that high quality mRNA can be isolated from a sorted population. Unfixed MDA-468 cells, a human breast cancer cell line, were temporarily permeabilized with lysolecithin. A range of lysolecithin concentrations were studied in order to optimize DNA staining but minimize alterations in cell size and integrity. Cells permeabilized with lysolecithin in PBS, were stained with propidium iodide (50 pg/ml) in the presence of 1% bovine serum albumin, 1 mM Na,EDTA in PBS. The optimal concentration (4 pg lysolecithin/ml) combined staining -90% of the cells for DNA, with minimal effects on cell size. Subsequently, the cells were assayed for DNA content as a measure of cell cycle position. MDA-468 cells identified as being in G1 were sorted and collected. From these, high quality mRNA could be isolated, as judged by its ability to be in vitro translated into protein of a wide range of molecular weights. This technique should be useful for molecular studies requiring discrete cell populations based on DNA content andlor cell Cycle position. 0 1993 Wiley-Liss, Inc.Key terms: Flow cytometry, MDA-468, lysolecithin permeabilization, DNA staining, cell integrity, cell cycle, in vitro translationThe examination of DNA content by flow cytometric analysis has been widely used to investigate the ploidy status, DNA index, and cell cycle phase percentages of cells and nuclei from tissue and cell samples. Several fluorescent staining methods have been developed for the analysis and sorting of individual cells according to their DNA content. However, many of the common nonvital dyes used to stain cellular DNA quantitatively are efficiently excluded from intact plasma membranes. In contrast, some more recently introduced vital dyes are taken up by living cells. However, most have excitation wavelengths below 460 nm, limiting their use to lasers with UV capability. Therefore, preparative techniques that enable one to identify and collect a particular cell cycle fraction from heterogeneous cell populations using the newer air-cooled argon lasers would be useful. This is particularly true for studying gene expression correlated with differentiation and cell cycle status.We report a permeabilization technique using lysolecithin that allows DNA to be stained with dyes normally excluded from viable cells while maintaining the integrity of the cell sufficiently to allow for the isolation of high quality mRNA. Human breast cancer cells (MDA-468) were permeabilized with low concentrations of lysolecithin, stained for DNA using propidium iodide (PI), and analyzed for cell cycle kinetics. Cells in G1 of the cell cycle were identified and sorted. From these G1 sorts, high quality mRNA that could be efficiently translated into proteins of a wide range of molecular ...

Flow sorting from organ material by intracellular markers

“…To our knowledge, very few studies have described sorting of permeabilized cells for molecular studies (1)(2)(3)14,15). Importantly, most of the published studies have utilized cell cultures (as opposed to organ material), or utilized DNA staining only (as opposed to immunostaining for intracellular antigen) (1,2,14,15), or performed PCR (as opposed to RT-PCR) (16).…”

“…In summary, our laboratory is interested in developing generally applicable methods for sorting rare cell subsets from organ material based on intracellular markers, for molecular toxicology studies. To our knowledge, very few studies have described sorting of permeabilized cells for molecular studies (1)(2)(3)14,15). Importantly, most of the published studies have utilized cell cultures (as opposed to organ material), or utilized DNA staining only (as opposed to immunostaining for intracellular antigen) (1,2,14,15), or performed PCR (as opposed to RT-PCR) (16).…”

“…To our knowledge, very few studies have described sorting of permeabilized cells for molecular studies (1)(2)(3)14,15). Importantly, most of the published studies have utilized cell cultures (as opposed to organ material), or utilized DNA staining only (as opposed to immunostaining for intracellular antigen) (1,2,14,15), or performed PCR (as opposed to RT-PCR) (16). Barrett and coworkers sorted epithelial cells from mammary biopsy specimens based on cytokeratin staining, but did not attempt to sort rare cell subsets, and only RT-PCR analysis was performed on the sorted cells (3).…”

Miniaturization in functional genomics and proteomics

“…Techniques such as cell sorting 11 , manual dissection 12 and LASER CAPTURE MICRODISSECTION (LCM) 13 important prerequisite in many biological studies. However, even cells of the same type usually represent different stages of physiological processes.…”

Separation of live cells in different phases of the cell cycle for gene expression analysis

“…Methods for the physical separation of cells based on DNA content have been developed (2)(3)(4)(5)(6)(7)(8). DNA staining of viable cells followed by flow cytometric analysis and sorting provides an ideal alternative approach for selecting and recovering cells from various phases of the cell cycle.…”