Isolation of full‐size mRNA from ethanol‐fixed cells after cellular immunofluorescence staining and fluorescence‐activated cell sorting (FACS)

Abstract

Preparation of intact, full-size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fmatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, fullsize RNA suitable for Northern blotting from cells that were fixed by 95% ethanoY5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS. o 1995 wiley-Liss, Inc.

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

“…Diez et al (16) reported isolating RNA after using short ethanol fixation and cell sorting. However, this technique can lead to a time-dependent increase in loss of macromolecules through pores in the cell membrane and to rapid nucleic acid degradation by active or insufficiently inactivated ribonucleases (17). The present data regarding cyclin B1 expression levels demonstrated that this methodology generates confirmatory results.…”

“…Cell fixation, required for specific identification of populations to be sorted, may result in RNA degradation (16). In fact, routinely used fixatives such as ethanol and formaldehyde do not completely inactivate RNAses (17). In the present report, we described a new approach to separate cell cycle populations by fluorescence-activated cell sorter from live cells without cell fixation, followed by isolation of full-length mRNA from sorted cells.…”

Flow sorting from organ material by intracellular markers

“…Thus, sorting from organ material based on intracellular markers would in many cases be an attractive option. Unfortunately, the fixation and permeabilization required for intracellular staining can be associated with RNA degradation (1)(2)(3).…”

“…Reflecting the technical difficulties, only few studies have described molecular analysis of cells flow-sorted for intracellular protein markers (1)(2)(3). Most work has been done on cell lines (1,2), with only one study describing flow sorting from dissociated biopsy specimens (3).…”

“…Most work has been done on cell lines (1,2), with only one study describing flow sorting from dissociated biopsy specimens (3). In that study, an abundant cell population was sorted (epithelial cells from mammary biopsies), and only RT-PCR was performed on the sorted cells (3).…”