Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.     

迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究

迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。     

Specific termination of RNA polymerase synthesis as a method of RNA and DNA sequencing.

Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells.Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.     

Cytochemical hybridisation with fluorochrome-labelled RNA

Summary A new method to localise specific DNA sequences in microscopic preparations by hybridocytochemistry using fluorochrome labelled complementary RNA has been described recently (Bauman et al. 1981). The present paper describes a procedure to increase the sensitivity of this method. RNA complementary to kinetoplasts DNA of Crithidia luciliae was labelled with fluorescein and hybridised with Sephadex beads to which kinetoplast DNA or heterologous DNA had been covalently bound as well as to Crithidia luciliae preparations. The fluorescein-labelled RNA was found to hybridize specifically with homologous DNA both on the beads and in the cells. The sensitivity of the hybrid detection could be increased by applying an indirect immunofluorescence reaction using rabbit antiserum raised against the hapten fluorescein as has been described for the amplification of a direct immunofluorescence reaction by Schmitz and Kampa (1979). The complete procedure resulted in an amplification of the original specific fluorescence both on the beads and in the cells. The increase was quantified by microfluorimetry. Several aspects of the immunocytochemical amplifying reaction were quantitatively investigated using Sephadex beads to which poly(A) or DNA was coupled and FITC-labelled poly(U) or cRNA was hybridised. A 5- to 10-fold amplification was obtained both in the beads and on the cell preparations. When the amplifying steps were repeated a proportional increase in background fluorescence was observed.This work was supported by the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.)     

Analysis of RNA flexibility by scanning force spectroscopy

Scanning force spectroscopy was used to measure the mechanical properties of double stranded RNA molecules in comparison with DNA. We find that, similar to the B–S transition in DNA, RNA molecules are stretched from the assumed A′ conformation to a stretched conformation by applying a defined force (plateau force). The force depends on the G + C content of the RNA and is distinct from that required for the B–S transition of a homologous DNA molecule. After the conformational change, DNA can be further extended by a factor of 0.7 ± 0.2 (S-factor) before melting occurs and the binding of the molecule to the cantilever is finally disrupted. For RNA, the S-factor was higher (1.0 ± 0.2) and more variable. Experiments to measure secondary structures in single stranded RNA yielded a large number of different force-distance curves, suggesting disruption and stretching of various secondary structures. Oriented attachment of the molecules to the substrate, a defined pick-up point and an increased resolution of the instrument could provide the means to analyse RNA secondary structures by scanning force spectroscopy.     

Synthesis of RNA using T7 RNA polymerase and immobilized DNA in a stream type reactor]

The DNA ligase-induced assembly of synthetic oligodeoxyribonucleotides on polymer supports was used to obtain immobilized DNA, containing the T7 RNA polymerase promoter and coding for a 14-membered oligoribonucleotide. The obtained template can carry out RNA synthesis in a flowing-type reactor. Sepharose 4B and Toyopearl HW-55 were used as supports.     

Simultaneous banding of rat embryo DNA, RNA, and protein in cesium trifluoroacetate gradients

A procedure for the simultaneous banding of cellular DNA, RNA, and protein by centrifugation in cesium trifluoroacetate (CsTFA) gradients is described. Starting with homogenates of Day 11 rat embryos, this procedure was used to separate total DNA, RNA, and protein. Under the conditions used DNA banded at a peak density of 1.63 g/ml, RNA at a peak density of 1.83 g/ml, and protein at a peak density of 1.40 g/ml. Nucleic acids isolated from CsTFA gradients were judged to be protein free. RNA isolated by this method is apparently free of DNA contamination; however, DNA isolated by this method does contain some RNA (less than 5% contamination).