Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 10948434

Control selection for RNA quantitation.

T Suzuki·P J Higgins·D R Crawford
View original source ↗Export metadata ↓

Abstract

The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of interest under different conditions such as stress, growth, development, cell and tissue localization or as part of an evaluation of the effects of gene transfection. A variety of techniques exist to measure gene expression and most commonly involve Northern hybridization analysis, ribonuclease protection or RT-PCR. Common to all of these assays is the inclusion of a so-called loading or internal control (i.e., analysis of an mRNA that does not change in relative abundance during the course of treatments). Here, we discuss the uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, with special emphasis on precautions associated with the use of GAPDH.

Explore related subjects

ActinsAnimalsCalcitriolCells, CulturedCricetinaeCytokinesDNA, RibosomalEnzyme InductionGlyceraldehyde-3-Phosphate DehydrogenasesHeat-Shock ProteinsHormonesHot TemperatureHumansManganeseMesocricetusMiceMice, Inbred BALB COrgan SpecificityOxidative StressRNA, MessengerRNA, RibosomalRatsResearch DesignTranscription, GeneticTubulinTumor Cells, CulturedComparative StudyJournal ArticleResearch Support, U.S. Gov't, P.H.S.ReviewValidation Study

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

T Suzuki, P J Higgins, D R Crawford. 2000. Control selection for RNA quantitation.. https://doi.org/10.2144/00292rv02

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2009

Nap1-mediated actin remodeling is essential for mammalian myoblast fusion.

Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.

Actins↗
PubMed2002

Low-affinity CCK-1 receptors inhibit bombesin-stimulated secretion in rat pancreatic acini--implication of the actin cytoskeleton.

EXPERIMENTAL OBJECTIVES: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. RESULTS: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10(-10) M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125(FAK)) and a marked reorganisation of the actin cytoskeleton. CONCLUSIONS: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors.

Actins↗
PubMed2002

Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.

Actins↗