Analytical gel electrophoresis of high-molecular-weight RNA in acrylamide-agarose gels containing methylmercuric hydroxide

Sebo, Thomas J.; Schmit, Joseph C.

doi:10.1016/0003-2697(82)90328-1

Cited by 9 publications

(3 citation statements)

References 25 publications

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“…Poly(A) + RNA was prepared from different tissues of C57BL/6J mice and SFME cell lines using mRNA isolation kit (Roche Diagnostics, Quebec, Canada) and analysed by Northern blot hybridization using 2.2% (w/v) acrylamide-0.5% (w/v) agarose gel [63]. The blot was probed with a 32 P-labelled 3.3 Kb RNA probe generated from Dlc1 6.1 Kb isoform.…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

et al. 2010

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BackgroundThe Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.ResultsIn addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay.ConclusionsThe mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.

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Section: Methodsmentioning

confidence: 99%

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

et al. 2010

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“…Composite gels have been mainly used for the analysis of nucleic acids (ssRNA 127, 128, DNA 129) and nucleic acid−protein complexes (nucleosomes 130, spliceosomes 131, viruses 132). Applications to proteins typically deal with SDS‐PAGE (see Section 5.2).…”

Section: Analysis Of High Molecular Size Proteinsmentioning

confidence: 99%

Other than IPG‐DALT: 2‐DE variants

2010

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Special problems require special solutions. That is why, despite the trend towards standardization of the procedures in proteomic investigations, a number of alternative protocols, based on 2-DE, have been devised with time and are applied to the resolution of proteins on which the performance of IPG-DALT is poor. The difficult-to-handle samples include high pI, high molecular size and high hydrophobicity proteins. The protocol variants entail changes in the gel media and/or the use of unusual additives (often surfactants or solvents). But also: Specific questions require specific answers. When the aim is not the resolution of individual polypeptide chains in a fully unfolded state but the analysis of higher-order structures (proteins assembled from subunits, complexes assembled from interacting proteins) the standard conditions under which IPG-DALT is performed turn inadequate. Electrophoresis is then performed either in first dimension or in both first dimension and second dimension under non-denaturing (native) and/or non-reducing conditions.

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“…A variety of approaches have been developed centered around gel electrophoresis under denaturing conditions, with the aim of disrupting RNA secondary structure to provide accurate molecular weight determinations (Sambrook et al 1989). Many of the current methodologies utilizing denaturing conditions incorporate the use of toxic, hazardous chemicals such as glyoxal/dimethyl sulfoxide and methyl mercury hydroxide (Bailey and Davidson 1976;McMaster and Carmichael 1977;Sebo and Schmit 1982). Alternative denaturants include urea, formamide, and formaldehyde (Floyd et al 1974;Pinder et al 1974;Lehrach et al 1977).…”

Section: Introductionmentioning

confidence: 99%

Enrichment and analysis of RNA centered on ion pair reverse phase methodology

Dickman¹,

Hornby²

2006

RNA

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Here we describe a procedure for the rapid enrichment of RNA from cell extracts and the subsequent fractionation and analysis of the ''small RNA'' population by ion pair reverse phase chromatography. Solid phase extraction procedures have been developed utilizing nonporous alkylated poly(styrene-divinylbenzene) particles in conjunction with ion pair reagents to enrich total RNA. This approach facilitates the selective enrichment and separation of the relatively lower abundance small RNAs, from the more abundant higher molecular weight rRNA species. We also describe the application of monolithic capillaries in conjunction with ion pair reverse phase chromatography to bring increased sensitivity in the analysis of very low abundance RNAs. These approaches will simplify the biochemical analysis of this class of molecules, which are emerging as important regulators of global gene expression in higher organisms.

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Analytical gel electrophoresis of high-molecular-weight RNA in acrylamide-agarose gels containing methylmercuric hydroxide

Cited by 9 publications

References 25 publications

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

Other than IPG‐DALT: 2‐DE variants

Enrichment and analysis of RNA centered on ion pair reverse phase methodology

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