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      Guidelines for Experiments Using Antisense Oligonucleotides and Double-Stranded RNAs

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          Abstract

          After decades of research and development, synthetic nucleic acids are beginning to enjoy significant success in the clinic. Approved drugs have increased interest in the field, and many basic research studies have focused on synthetic nucleic acids to control the action of messenger RNA and noncoding RNAs. Unfortunately, experimental designs are often inadequate, resulting in misleading interpretation of data and unconvincing work that wastes resources and does little to advance the field. The goal of this commentary is to outline the problems facing many researchers, especially those new to the use of synthetic oligonucleotides. We describe the minimum control experiments necessary to build a strong case for real effects that are likely due to interactions at the intended molecular target. A common set of standards for preparing and judging experiments should facilitate better interpretation of data and publications that contribute positively to using synthetic nucleic acids as tools and drugs.

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          Most cited references22

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          RNAi Factors are Present and Active in Human Cell Nuclei

          Summary RNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP and TRNC6A/GW182 are in the human nucleus and associate together in multi-protein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus.
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            Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents

            For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
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              With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies

              The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.
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                Author and article information

                Journal
                Nucleic Acid Ther
                Nucleic Acid Ther
                nat
                Nucleic Acid Therapeutics
                Mary Ann Liebert, Inc., publishers (140 Huguenot Street, 3rd FloorNew Rochelle, NY 10801USA )
                2159-3337
                2159-3345
                01 June 2019
                30 May 2019
                30 May 2019
                : 29
                : 3
                : 116-122
                Affiliations
                [ 1 ]Department of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University, Carbondale, Illinois.
                [ 2 ]Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois.
                [ 3 ]Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, Texas.
                Author notes
                [*]Address correspondence to: David R. Corey, PhD, Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, 6001 Forest Park, Dallas, TX 75390 david.corey@ 123456utsouthwestern.edu
                Article
                10.1089/nat.2018.0772
                10.1089/nat.2018.0772
                6555184
                30907681
                ce8d9ad9-bed4-4e93-89c7-d839573740e6
                © Keith T. Gagnon and David R. Corey, 2019; Published by Mary Ann Liebert, Inc.

                This Open Access article is distributed under the terms of the Creative Commons License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 12 December 2018
                : 05 February 2019
                Page count
                Figures: 3, Tables: 1, References: 28, Pages: 7
                Categories
                Issues in Development

                antisense oligonucleotide,duplex rna,controls,guidelines

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