Large and Small Scale RNA Preparations from Eukaryotic Cells

Department of Molecular and Tumor Therapy, Max-Delbruck-Centre for Molecular Medicine, Berlin, Germany.
Methods in Molecular Biology (Impact Factor: 1.29). 02/1998; 86:7-14. DOI: 10.1385/0-89603-494-1:7
Source: PubMed


A mammalian cell contains approx 10−5 μg of RNA which consists mainly of rRNA and in smaller amounts of a variety of low-mol-wt RNA species. These RNAs are of
defined size and sequence. The ability to isolate clean, intact, and DNA-free RNA is a prerequisite in analyzing gene expression
and cloning genes. The regulation of gene expression, e.g., the analyses of detailed function of transcription factors, promoter
and enhancer sequences, and RNA synthesis and processing as well as the analyses of gene expression after transfer of genes
of interest into eukaryotic cells are common areas of investigation in molecular biology. Important for the study of regulation
of gene expression is the ability to isolate, analyze, and quantify RNA molecules, specifically mRNAs coding for proteins
of interest. Furthermore, RNA is needed in order to copy it into double-stranded DNA for cloning and production of a cDNA
library. The critical first step in the construction of a cDNA library is the efficient isolation of undegraded total RNA.
The major difficulty in RNA isolation is the presence of ribonucleases found in virtually all tissues and liberated either
during cell lysis or accidentally introduced in traces from other potential sources.

6 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Measuring RNA turnover is important because of the significance of rRNA, tRNA and mRNA in tissue protein synthesis. Changes in turnover of each of these species precede important cellular events such as hormone or cytokine action or cell-division itself. Isotopic methods have relied on decay of pulse-labelled RNA or on incorporation of isotopically-labelled precursors. However, recycling of labels may lead to under or overestimation of synthesis rates respectively. The labelling of the intracellular precursor pool must be known if accurate RNA synthesis rates are to be calculated from the degree of incorporation. However, the intracellular nucleotide pools may be anatomically or metabolically compartmented (i.e. via de novo or salvage synthesis routes) and this complicates many study designs. The use of[methyl-14C]- or [methyl-3H]methionine as a means of labelling methylated nucleosides in RNA and protein simultaneously is described in addition to new stable isotopic techniques based on 13C-glycine as a de novo label. Urinary excretion of the numerous modified nucleosides in cellular RNA can be used to calculate whole-body turnover rates of each of the major RNA species. Examples of the effects of critical-illness and glutamine supplementation on RNA turnover are given. We conclude by suggesting that whole-body RNA turnover rates have been significantly underestimated and that this has implications for nutritional therapy, especially with regard to nucleotide supplementation.
    No preview · Article · Oct 2000 · Current Opinion in Clinical Nutrition and Metabolic Care
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.
    Full-text · Article · Nov 2001 · American Journal Of Pathology