Detection of mRNA in flow‐sorted cells

Dunne, John F.; Jan, Thomas; Lee, S.

doi:10.1002/cyto.990100211

Cited by 7 publications

(3 citation statements)

References 22 publications

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“…The sort blot technique was performed with modifications of the method previously described (Dunne et al, 1989). Changes in the method included modifications in the sort blot module design and the use of nylon membranes instead of nitrocellulose.…”

Section: Sort Blotmentioning

confidence: 99%

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon‐γ and interleukin‐1α

Quintavalla¹,

Berg

Beavis

et al. 1993

Journal Cellular Physiology

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Articular chondrocytes from rheumatoid joints have been shown to express class II major histocompatibility (MHC) antigens that were correlated with the presence of interferon-gamma (IFN-gamma) in the inflamed joint. Chondrocytes expressing MHC antigens function as antigen presenting cells and thus stimulate lymphocyte proliferation. These responses suggest a powerful role for the IFN-gamma stimulation of chondrocytes. The present studies were designed to examine the functional role of chondrocytes exposed to IFN-gamma during cartilage degradation that occurs in synovial disease. Destruction of cartilage in arthritis is partially attributable to metalloproteinases released by the chondrocytes in response to interleukin-1 (IL-1). Bovine articular chondrocytes treated with interleukin-1 alpha (IL-1 alpha) produced enhanced levels of stromelysin mRNA, however, Northern blots could not determine the percentage of cells responding. Exposure of bovine articular chondrocytes to IFN-gamma induced the expression of bovine HLA-DR (boHLA-DR) antigen in 50% of the cells. Using a modified cell sorting technique, chondrocytes that expressed class II MHC antigens produced two fold greater stromelysin mRNA than chondrocytes that did not express this antigen. In contrast, collagen type II mRNA levels were similar in chondrocytes, regardless of the expression of class II MHC antigens. In situ hybridization studies showed that less than half of all cartilage chondrocytes were induced to synthesize stromelysin mRNA. These observations suggest that IFN-gamma stimulates specific subpopulations of chondrocytes to be functionally active in inflammation-induced metalloprotease secretion.

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Section: Sort Blotmentioning

confidence: 99%

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon‐γ and interleukin‐1α

Quintavalla¹,

Berg

Beavis

et al. 1993

Journal Cellular Physiology

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“…Several reports have shown that intact RNA can be recovered from cells flow-sorted after labeling of cytoplasmic epithelial cell-specific cytokeratins (2,4,7,8). Whole-cell sorting typically involves treating cells in organic fixatives, followed by staining with tissuespecific antibodies in an aqueous solution.…”

Section: Introductionmentioning

confidence: 99%

High-Quality RNA and DNA from Flow Cytometrically Sorted Human Epithelial Cells and Tissues

et al. 2002

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Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater, which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality afterflow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissues.

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“…They flow-sorted living cells directly onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After hybridization of these filters with 32p-labeled specific DNA probes, they were able to study the phorbol ester/lipopolysaccharide induction of IL-1 mRNA in U937 cells, and they demonstrated the linearity of the method by exploiting the abundant expression of the c-los in these cells [15]. IL-I mRNA was readily detectable autoradiographically from as few as 5 000 induced cells, and liquid scintillation counting demonstrated a good linearity in c-fos quantification over the range of 1 000 to 60 000 cells sorted per filter.…”

Section: Specific Mrna Quantificationmentioning

confidence: 99%

Flow cytometry and RNA studies

Grünwald

1993

Biology of the Cell

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RNA was less studied by flow cytometry (FCM) in comparison to other cellular components. Moreover, most of these studies were applied to the measurement of total RNA. Nevertheless, such a quantification, essentially by the use of acridine orange, but also other dyes or dye combinations, permitted the acquisition of important information about the general metabolism, and the characterization of different tumoral cell types. More recently, FCM has been used to quantify specific mRNA, in two different approaches: the first one employs the sorting followed by RNA extraction and/or detection by specific radiolabeled probes; in the second approach, the use of fluorescence in situ hybridization (FISH) on cells in suspension allowed the direct quantification of specific mRNA by FCM.

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Detection of mRNA in flow‐sorted cells

Cited by 7 publications

References 22 publications

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon‐γ and interleukin‐1α

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon‐γ and interleukin‐1α

High-Quality RNA and DNA from Flow Cytometrically Sorted Human Epithelial Cells and Tissues

Flow cytometry and RNA studies

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