Degradation Of Ribonucleic Acid Research Articles

Antibody detection of translocations in Ewing sarcoma

The detection of chromosomal translocations has important implications in the diagnosis, prognosis and treatment of patients with cancer. Current approaches to translocation detection have significant shortcomings, including limited sensitivity and/or specificity, and difficulty in application to formalin-fixed paraffin-embedded (FFPE) clinical samples. We developed a new approach called antibody detection of translocations (ADOT) that avoids the shortcomings of current techniques. ADOT combines a transcriptional microarray-based approach with a novel antibody-based detection method. ADOT allows for the accurate and sensitive identification of translocations and provides exon-level information about the fusion transcript. ADOT can detect translocations in poor-quality unprocessed total ribonucleic acid (RNA). Furthermore, the technique is readily generalizable to detect any potential fusion transcript, including previously undescribed fusions. We demonstrate the feasibility of ADOT by examples in which both known and unknown Ewing sarcoma translocations are identified from cell lines, tumour xenografts and FFPE primary tumours. These results demonstrate that ADOT may be an effective approach for translocation analysis in clinical specimens with significant RNA degradation and may offer a novel diagnostic tool for translocation-based cancers.

Comparative study of different protocols for isolation of RNA from polysaccharides and polyphenolics rich plant tissues

The isolation of high quality ribonucleic acid (RNA) from plant has been a difficult problem due to presence of high amount of polysaccharides, polyplenolics and nucleases that mediate RNA degradation. After comparative analysis of four popular protocols for RNA isolation, a modified method was developed which gave maximum yield with purity. Modified method is not only suitable for the isolation of intact RNA from immature seeds of legumes, containing high amount of starch, polyphenolics and several nucleases but also works successfully with other plant organs like leaves, epicotyls and callus. The yield of total RNA extracted by this method varies from 71 to 417mg/g tissues. Key words: RNA isolation, polysaccharides and polyphenolics rich plant tissues, pegionpea and immature seeds.

Sampling and RNA quality for diagnosis of honey bee viruses using quantitative PCR

Molecular diagnoses of pathogens via ribonucleic acid (RNA) signatures are used widely in honey bee pathology. Such diagnoses can be compromised by ubiquitous and endogenous RNA-degrading enzymes activated after the death of sampled bees. RNA degradation can be minimized by storage at ultra-cold temperatures or by immersion in high-salt buffers. However, these methods are not always available in the field or are costly, driving a search for alternative methods to store and transport bees for RNA analyses. While the impact of storage conditions on RNA integrity has been evaluated, the tolerance of standard RT-qPCR diagnostic methods of honey bee pathogens for suboptimal collection and storage is unknown. Given the short regions of RNA used for pathogen diagnosis (generally amplified regions of 100–200 nucleotides), it is conceivable that even degraded RNA will provide a template for precise diagnosis. In this study, the impact of the two most convenient sample storage and handling methods (+4°C and ambient temperature) for identifying honey bee virus infections was evaluated by RT-qPCR. The aim was to streamline the methods needed to collect, transport, and store honey bee samples destined for pathogen diagnosis. The data show that samples held at room temperature for times anticipated for sample transport for up to 5 days are suitable for diagnosis of two of the most common and prevalent honey bee viruses, deformed wing virus (DWV) and black queen cell virus (BQCV). The results will be useful for the standardisation of sampling methods across countries and laboratories.

Preservation of fine‐needle aspiration specimens for future use in RNA‐based molecular testing

The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues. FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif). Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas. FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies.

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues

The use of ribonucleic acid (RNA) extracted from Hepes glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixed tissues in quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is fairly novel. We compared qRT-PCR analysis of formalin- and HOPE-fixed, paraffin-embedded lymph node tissues from Mycobacterium bovis-infected cattle by extracting total RNA using a commercial kit (Ambion) and a Trizol method. RNA extracted from HOPE-fixed tissues showed comparable quantities between the commercial kit (82.7–107.9 μg/ml total RNA) and the Trizol method (87–161.1 μg/ml total RNA), displaying a high degree of integrity when analyzed by electrophoresis. RNA extracted from formalin-fixed tissues using the commercial kit produced similar concentrations (80.6–145.7 μg/ml total RNA) in comparison to the HOPE tissue; however, the integrity was compromised. Extraction of RNA from the formalin-fixed tissues using Trizol was unsuccessful. Following qRT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total RNA from HOPE-fixed tissues showed higher levels of target messenger ribonucleic acid (mRNA) (4.05×10 −2 pg/100 ng total RNA using the commercial kit and 6.45×10 −2 pg/100 ng total RNA using Trizol) in comparison to formalin-fixed tissues (5.69×10 −4 pg/100 ng total RNA). This could be attributed to RNA degradation by exposure to formalin fixation. In conclusion, the HOPE fixative proved to be a better source for RNA extraction from cattle lymph nodes and subsequent qRT-PCR.

Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.

SMN Transcript Stability: Could Modulation of Messenger RNA Degradation Provide a Novel Therapy for Spinal Muscular Atrophy?

Proximal spinal muscular atrophy is caused by deletion or mutation of the survival motor neuron 1 gene, SMN1. Rentention of a nearly identical copy gene, SMN2, enables survival but is unable to fully compensate for the loss of SMN1. The SMN1 and SMN2 genes differ by a single nucleotide that results in alternative splicing of SMN2 exon 7 due to the disruption of a binding site for an essential splicing factor. This alternatively spliced form encodes a partially functional truncated protein. Because SMN2 is present in patients with spinal muscular atrophy, it is an ideal therapeutic target. Some of the current approaches to increase SMN protein levels are aimed at increasing the transcription from SMN2 or at preventing exon 7 skipping. One area that has yet to be investigated is the stability of messenger ribonucleic acid (RNA) transcripts produced from SMN2. We postulated that transcripts derived from SMN2 may be less stable because alternative splicing, recruitment of RNA-binding proteins, and alteration of stop codons have been associated with changes in rates of messenger RNA decay; these features are all characteristic of SMN2. Accordingly, transcript degradation was examined within primary fibroblast cells that exclusively contained SMN1 or SMN2 by treating cultures with a transcriptional inhibitor to observe messenger RNA stability. The results indicate that SMN transcript instability does not play a role in the disease mechanism, suggesting that therapeutic modulation of messenger RNA degradation would not target a molecular defect in patients with spinal muscular atrophy, although it could provide general benefits by increasing total pools of SMN2 transcripts.

Optimization of Ribonucleic Acid Detection From Archival Guinea Pig Temporal Bone Specimens

The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Natural and engineered ribonucleases as potential cancer therapeutics

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.

Characterization of the ribonuclease activity on the skin surface

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin® although not by the ribonuclease inhibitor SUPERase· In™. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development.

Silence of the transcripts: RNA interference in medicine

Silencing of gene expression by ribonucleic acid (RNA), known as RNA interference (RNAi), is now recognized as a major means of gene regulation in biology. In this mechanism, small noncoding double-stranded RNA molecules knock down gene expression through a variety of mechanisms that include messenger RNA (mRNA) degradation, inhibition of mRNA translation, or chromatin remodeling. The posttranscriptional mechanism of RNAi has been embraced by researchers as a powerful tool for generating deficient phenotypes without mutating the gene. In parallel, exciting recent results have promised its application in disease therapy. This review aims to summarize the current knowledge in this area and provide a roadmap that may eventually launch RNAi from the research bench to the medicine chest.

Ozone degradation of ribonucleic acid (RNA)

RNA (ribonucleic acid) from Torula utilis was subjected to ozonization both in aqueous solution and in the solid state. The ozonization in aqueous solution was followed by spectrophotometry and polarimetry. The latter technique was extremely useful in following the RNA degradation as function of the amount of O 3 added to the solution. The RNA specific optical rotation drops to zero following a linear relationship with the amount of O 3 added to the solution. The loss in the chirooptical properties of RNA and the reduction of the specific viscosity of the solution were interpreted in terms of denaturation of the RNA macromolecule and in terms of oxidation of the purine and pyrimidine bases which are the pendent groups of RNA macromolecule. The polarimetric measurement of the reaction between O 3 and RNA has also allowed the measurement of the stoichiometric molar ratio at which the RNA optical rotation vanishes completely. This RNA/O 3 molar ratio was found to lie in the range of 2.0–1.5, suggesting that the RNA denaturation is complete when at least 50% of the RNA monomeric units have reacted with O 3. The RNA/O 3 molar ratio was also checked iodometrically and found about 1.3. The differences between the polarimetric and iodometric stoichiometry are discussed. RNA ozonized in powder form in the solid state was found to be significantly less reactive than in the case of the aqueous solution. After 24 h ozonization in the solid state, RNA shows a certain reduction in the specific optical rotation (much less compared to RNA ozonized in aqueous solution at the equivalent nominal molar ratio) suggesting the occurrence of a certain degree of denaturation. The FT-IR spectra of the ozonized RNA are discussed in comparison to the reference pristine RNA spectrum.

Assay for evaluating ribonuclease H-mediated degradation of RNA-antisense oligonucleotide duplexes.

Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid (DNA)-RNA duplex substrates, and so may provide a unique mode of therapeutic intervention at the genetic level of virtually any disease. We have therefore set out detailed procedures for conducting routine assays with almost any one of this family of enzymes by a straightforward assay aimed at identifying novel enzyme-activating antisense oligonucleotides (AONs). The procedures described herein should enable easy identification of potent AON molecules, provided that the RNA is appropriately labeled for subsequent visualization following the guidelines set forth in this protocol.

Effect of triazole-type plant growth regulators on sunflower and safflower seed viability

Pretreatment of sunflower and safflower seeds with triazole-type plant growth regulators (triazoles) diminished the rate of germination and reduced seedling growth. However, if seeds were aged at 90% relative humidity (accelerated ageing), triazoles significantly reduced the deleterious effects of accelerated ageing on germination percentage and field emergence. Seedling vigour was also greater in seedlings raised from triazole-treated seeds than from untreated seeds after accelerated aging. The activities of enzymes like dehydrogenase declined at a faster rate during accelerated ageing in untreated control than in triazole-treated seeds. Triazoles also slowed down the degradation of ribonucleic acid. The rate of change of soluble carbohydrates and amino acids was low in triazole-treated seeds compared with untreated control seeds. A positive correlation was evident between increase in membrane permeability and loss of seed viability. Membrane permeability and level of malondialdehyde, a peroxidation product of unsaturated fatty acids, gradually increased in storage at 90% relative humidity, but in seeds pretreated with triazoles these changes were minimal. Key words: triazoles, accelerated ageing, sunflower and safflower seed viability.

核酸系調味料製造技術の発展

This paper reviews the progress in the technology for manufacturing 5'-inosinic and 5'-guanylic acids as flavor-enhancing agents. It is only past 25 years from the beginning of their commercial production. Today, there are two ways for their production : One is degradation of ribonucleic acid from yeast and the other, fermentation from sugars using bacterial mutants. It is difficult to say now which process is better, because both processes include some severe problems to solve for the further technological development.The keys for the further development of the degradation process are (a) availability of ribonucleic acid from methanol bacteria produced as single cell protein, (b) development of a bioreactor consisting of immobilized enzyme systems, and (c) evaluation of utility values of 5'-uridylic and 5'-cytidylic acid as the byproducts. The keys for the fermentation process are (a) increase in the productivity of nucleosides or 5'-nucleotides by strain improvement by means of gene engineering techniques, and (b) development of a bioreactor using immobilized cells. They are now on the way of research and development in the third stage of the progress in the technology for manufacturing flavor-enhancing 5'-ribonucleotides.

Indirect estimation of biomass by rapid ribonucleic acid determination

Microbial ribonucleic acid (RNA) was estimated by determinations of nucleotides formed by complete hydrolysis. The steps involved in this procedure comprise the alkaline hydrolysis of RNA (5 N NaOH; 100°C; 5 min) to a mixture of 2′-and 3′-mononucleotides and successive separation and quantification by means of high performance liquid chromatography (HPLC). Since guanylic acid (GMP) shows adequate stability during RNA degradation and may be separated well and rapidly from other compunds, it is used as an equivalent measure of RNA. The assay is performed within 15 min having a lowest detection limit of 4 mg RNA/l. It was applied to isolated yeast cells and fermentation broths of mycelial organisms. Linear correlation to mycelial dry weight was established for unlimited growth (trophophase), while in idiophase determination of RNA may serve as a measure of active biomass.

Glucocorticoids stimulate ribonucleic acid degradation in isolated rat thymic lymphocytes in vitro.

Glucocorticoids are known to stimulate lymphoid cell death, but little information is available regarding the biochemical mechanisms of adrenal steroid-induced lymphocytolysis. Previous work from our laboratory indicates that lymphocytes, which die in response to glucocorticoid, exhibit enhanced rates of cellular protein degradation. These findings encouraged us to study the turnover of other lymphocyte cellular macromolecules, namely RNA, as regulated by adrenal steroids. Thymic lymphocytes were pulse labeled in vitro in Eagle's Minimum Essential Medium supplemented with 10 μCi/ml [3H] uridine. Precursor incorporation into RNA was effectively stopped by a chase/dilution (1:20) of cells into fresh medium supplemented with 10 mM of both unlabeled uridine and cytidine. Cells were incubated in this medium for up to 4 h at 37 C with or without dexamethasone (Dex), and the remaining RNA-associated radioactivity was measured either by acid precipitation or with a multiple automated sample harvester. Control cel...

Codon-specific serine transfer ribonucleic acid degradation in avian liver during vitellogenin induction.

The relative rates of degradation of two major tRNASer species in rooster liver were simultaneously assessed during induction by estradiol-17 beta benzoate of the synthesis of a serine-rich phosphoprotein, vitellogenin. The relative rate of degradation was determined by an in vivo pulse-chase labeling method, which included a 24-h labeling period with [5-3H]orotic acid prior to and a 6-day chase period with nonradioactive orotic acid after the administration of estrogen. tRNA Ser(AGU,C) and tRNASer (UCU,C,A) were extensively purified by chromatography on benzoylated DEAE-cellulose in the presence and absence of Mg2+ and their radioactivities determined. In three separate labeling experiments, the difference in radioactivity of pulse-labeled and chased tRNASer (AGU,C) vs. that of tRNASer(UCU,C,A) was approximately 2-fold, suggesting a slower rate of degradation of tRNASer(AGU,C) during vitellogenin induction. Calculation of the approximate half-lives of the two tRNASer species indicates that the half-life of tRNASer (AGU,C) was increased from 3.1 days to 6.2 days during vitellogenin induction, while that of tRNASer (UCU,C,A) was essentially unchanged (2.6 days). Regulation of tRNA degradation which is possibly connected with the frequency of its use in ribosomal protein synthesis may help to explain why, in many differentiated cells, the tRNA population is adapted to the amino acid composition of the synthesized proteins.

Role of 2',5'-oligo(adenylic acid) polymerase in the degradation of ribonucleic acid linked to double-stranded ribonucleic acid by extracts of interferon-treated cells.

RNA covalently linked to double-stranded RNA (dsRNA) is preferentially degraded in extracts of interferon-treated HeLa cells [Nilsen, T. W., & Baglioni, C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2600-2604]. The size of the dsRNA required for this preferential degradation has been determined by annealing poly(I) of known length to the poly(C) tract of encephalomyocarditis virus (EMCV) RNA or by annealing poly(U) to poly(A) of known length of vesicular stomatitis virus mRNA. The dsRNA must be longer than about 60 base pairs to observe the preferential degradation of RNA. Moreover, triple-stranded regions that do not activate synthesis of 2',5'-oligo(A) and ethidium bromide, which intercalates in dsRNA and blocks 2',5'-olido(A) polymerase activation, prevent this degradation. Ethidium also blocks the degradation of the replicative intermediate of EMCV by extracts of interferon-treated cells. These experiments indicate that synthesis of 2',5'-oligo(A) is required for the degradation of RNA linked to dsRNA. The 2',5'-oligo(A)-dependent endonuclease does not cleave single- or double-stranded DNA, nor does it cleave homopolyribonucleotides. The potential role of the 2',5'-oligo(A) polymerase/endonuclease system in the inhibition of viral RNA replication is discussed.