Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells

Abstract

Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.

Inflammatory Monocytes Recruited to the Liver within 24 Hours after Virus-Induced Inflammation Resemble Kupffer Cells but Are Functionally Distinct

“…Another option is that both sorted cell types produce other IFN␣ subtypes then the IFN␣1 and IFN␣2 detected by the NanoString probes (NCBI accession numbers NM_010502.2 and NM_010503.2, respectively). Furthermore, due to the known RNA fragmentation after formalin fixation, all tested qPCR primer sets yielded inconsistent results when tested on RNA from sorted F4/80 high -Kupffer cells and inflammatory monocytes cells (data not shown) (35). IFN␤, on the other hand, was expressed by F4/80 high -Kupffer cells, but not by inflammatory monocytes, which may be the direct result of triggering of IFN-␤ production upon LCMV infection and repli- high -Kupffer cells.…”

“…After hybridization, transcripts were quantitated using the nCounter digital analyzer. Samples were run by the Johns Hopkins deep sequencing and microarray core facility (35). To correct for background levels, the highest negative-con-trol value for each sample was subtracted from each count value of that sample.…”

An Infant With Non-Ketotic Hyperglycemia: A Case Report

“…However, this type of preservation makes it difficult to perform molecular analysis on these type of samples due to nucleic acid modification by significant protein-protein and protein-nucleic acid crosslinks, following formalin/paraformaldehyde fixation. The fixation process itself doesn't cause nucleic acid fragmentation (12,13), instead, the embedding process, which requires high temperature and vacuum for the paraffin penetrance to the tissue, may lead to chemical reactions, which subsequently modify RNA and DNA. These modifications can cause fragmentation over time (14).…”

Selective depletion of cultured macrophages by magnetite nanoparticles modified with gelatin

“…However, the significance of cell-cell contact between macrophages and tumor cells remains unknown. Previous studies attempted to separate macrophages from tumor cells following direct co-culture experiments; however, it proved too difficult to separate the two types of cells (8,11). Sorting methods using flow cytometry were not suitable for gene expression analysis because of RNA degradation during the procedure (11).…”

“…Previous studies attempted to separate macrophages from tumor cells following direct co-culture experiments; however, it proved too difficult to separate the two types of cells (8,11). Sorting methods using flow cytometry were not suitable for gene expression analysis because of RNA degradation during the procedure (11). Although anti-cluster of differentiation (CD) 14 or CD11b antibody-labeled magnet beads are commonly available to rapidly isolate monocytes/macrophages, downregulation of CD14/CD11b has been observed on activated macrophages when using these beads (12,13).…”