A detergent-citric acid technique for isolating nuclear and cytoplasmic fractions containing undegraded RNA from cells of Xenopus laevis

Miller, Leon K.

doi:10.1016/0003-2697(79)90127-1

Cited by 12 publications

(4 citation statements)

References 20 publications

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“…To avoid any bias that may be due to selective damage of adherent cells in any phase of the cell cycle, the analysis was carried out on isolated nuclei rather than on detached cells. To this purpose, a protocol based on the citric acid method described by Miller [ 8 ] was used. Cells were seeded at 500,000 cells/ml on 6-well plates (Corning) or on non-adherent 6-well plates (suspension culture flasks from Greiner) and let to grow for 24 hours.…”

Section: Methodsmentioning

confidence: 99%

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

et al. 2021

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Except cells circulating in the bloodstream, most cells in vertebrates are adherent. Studying the repercussions of adherence per se in cell physiology is thus very difficult to carry out, although it plays an important role in cancer biology, e.g. in the metastasis process. In order to study how adherence impacts major cell functions, we used a murine macrophage cell line. Opposite to the monocyte/macrophage system, where adherence is associated with the acquisition of differentiated functions, these cells can be grown in both adherent or suspension conditions without altering their differentiated functions (phagocytosis and inflammation signaling). We used a proteomic approach to cover a large panel of proteins potentially modified by the adherence status. Targeted experiments were carried out to validate the proteomic results, e.g. on metabolic enzymes, mitochondrial and cytoskeletal proteins. The mitochondrial activity was increased in non-adherent cells compared with adherent cells, without differences in glucose consumption. Concerning the cytoskeleton, a rearrangement of the actin organization (filopodia vs sub-cortical network) and of the microtubule network were observed between adherent and non-adherent cells. Taken together, these data show the mechanisms at play for the modification of the cytoskeleton and also modifications of the metabolic activity between adherent and non-adherent cells.

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Section: Methodsmentioning

confidence: 99%

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

et al. 2021

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“…Citric acid disperses the cyto¬ plasmic components and inhibits the activity of RNases, both endogenous and exogenous (Miller, 1979). The NaDodS04 re¬ leases nucleic acids from their association with proteins.…”

Section: Methodsmentioning

confidence: 99%

Laboratory Methods

Dreier

Högger²,

Sorg³

1998

DNA and Cell Biology

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Total RNA was isolated from human leukocytes (monocytes, granulocytes), various cell lines (COS-7, Mono-Mac-6, L-132, HaCaT, EA.hy926, HL-60), and fungal mycelium by a rapid two-step method. Cells were lysed with NaDodSO4 in a citric acid-containing buffer. This procedure was succeeded by salt precipitation to remove contaminating DNA and protein and a final alcohol precipitation of RNA. Isolated RNA was of high quality, with a reasonable yield and little or no protein or DNA contamination. We present here a fast method for preparing RNA particularly from cell lines, which limits the use of toxic compounds.

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“…Cell fractionation and isolation of RNA Nuclear-cytoplasmic fractionation was performed as previously described (Miller, 1979) and RNA was isolated from the cell fractions using phenol and chloroform (Miller, 1978) or guanidine-HC1. Poly(A)'RNA was isolated using oligo d(T)-cellulose as described by Johnson et al (1974).…”

Section: Cell Culturementioning

confidence: 99%

“…Growing and resting cells were used to prepare nuclear and cytoplasmic fractions according to Miller (1979) and the cytoplasmic RNA content was determined using three different methods (Table 1). Although the RNA quantities measured varied according to the method of isolation, the ratio of RNA content of growing and resting cells was similar for all of the methods.…”

Section: Rna Content Of Growing and Resting X1-177 Cellsmentioning

confidence: 99%

Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis

Budorick

Miller

1982

Journal Cellular Physiology

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The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing the resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)+ mRNA by uniform labeling with 3H-uridine and by 3H-poly(U) hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.

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A detergent-citric acid technique for isolating nuclear and cytoplasmic fractions containing undegraded RNA from cells of Xenopus laevis

Cited by 12 publications

References 20 publications

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

A proteomic-informed view of the changes induced by loss of cellular adherence: The example of mouse macrophages

Laboratory Methods

Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis

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