Nonsense-Mediated Decay in Genetic Disease: Friend or Foe?

PubMed Central

Miller, Jake N.; Pearce, David A.

2014-01-01

Eukaryotic cells utilize various RNA quality control mechanisms to ensure high fidelity of gene expression, thus protecting against the accumulation of nonfunctional RNA and the subsequent production of abnormal peptides. Messenger RNAs (mRNAs) are largely responsible for protein production, and mRNA quality control is particularly important for protecting the cell against the downstream effects of genetic mutations. Nonsense-mediated decay (NMD) is an evolutionarily conserved mRNA quality control system in all eukaryotes that degrades transcripts containing premature termination codons (PTCs). By degrading these aberrant transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults, such as dominant negative effects or the ER stress response. Although NMD functions to protect the cell against the deleterious effects of aberrant mRNA, there is a growing body of evidence that mutation-, codon-, gene-, cell-, and tissue-specific differences in NMD efficiency can alter the underlying pathology of genetic disease. In addition, the protective role that NMD plays in genetic disease can undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring nonsense mutations. Here, we review the normal function of this RNA surveillance pathway and how it is regulated, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and how NMD can be used as a therapeutic target. PMID:25485595

Preparation and Analysis of RNA Crystals

NASA Technical Reports Server (NTRS)

Todd, Paul

2000-01-01

The crystallization of RiboNucleic Acids (RNA) was studied from the standpoint of mechanisms of crystal growth in three tasks: (1) preparation of high-quality crystals of oligonuclotides for X-ray diffraction, (2) finding pathways to the growth of high-quality crystals for X-ray diffraction and (3) investigation of mechanisms of action of inertial acceleration on crystal growth. In these tasks: (1) RNA crystals were prepared and studied by X-ray diffraction; (2) a pathway to high-quality crystals was discovered and characterized; a combination of kinetic and equilibrium factors could be optimized as described below; and (3) an interplay between purity and gravity was found in a combination of space and ground experiments with nucleic acids and proteins. Most significantly, the rate of concentration of precipitant and RNA can be controlled by membrane-based methods of water removal or by diffusion of multivalent cations across an interface stabilized by a membrane. Oligonucleotide solutions are electrokinetically stabilized colloids, and crystals can form by the controlled addition of multivalent cations.

Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons.

PubMed

Fritzsche, Renate; Karra, Daniela; Bennett, Keiryn L; Ang, Foong Yee; Heraud-Farlow, Jacki E; Tolino, Marco; Doyle, Michael; Bauer, Karl E; Thomas, Sabine; Planyavsky, Melanie; Arn, Eric; Bakosova, Anetta; Jungwirth, Kerstin; Hörmann, Alexandra; Palfi, Zsofia; Sandholzer, Julia; Schwarz, Martina; Macchi, Paolo; Colinge, Jacques; Superti-Furga, Giulio; Kiebler, Michael A

2013-12-26

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Tissue-aware RNA-Seq processing and normalization for heterogeneous and sparse data.

PubMed

Paulson, Joseph N; Chen, Cho-Yi; Lopes-Ramos, Camila M; Kuijjer, Marieke L; Platig, John; Sonawane, Abhijeet R; Fagny, Maud; Glass, Kimberly; Quackenbush, John

2017-10-03

Although ultrahigh-throughput RNA-Sequencing has become the dominant technology for genome-wide transcriptional profiling, the vast majority of RNA-Seq studies typically profile only tens of samples, and most analytical pipelines are optimized for these smaller studies. However, projects are generating ever-larger data sets comprising RNA-Seq data from hundreds or thousands of samples, often collected at multiple centers and from diverse tissues. These complex data sets present significant analytical challenges due to batch and tissue effects, but provide the opportunity to revisit the assumptions and methods that we use to preprocess, normalize, and filter RNA-Seq data - critical first steps for any subsequent analysis. We find that analysis of large RNA-Seq data sets requires both careful quality control and the need to account for sparsity due to the heterogeneity intrinsic in multi-group studies. We developed Yet Another RNA Normalization software pipeline (YARN), that includes quality control and preprocessing, gene filtering, and normalization steps designed to facilitate downstream analysis of large, heterogeneous RNA-Seq data sets and we demonstrate its use with data from the Genotype-Tissue Expression (GTEx) project. An R package instantiating YARN is available at http://bioconductor.org/packages/yarn .

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

RNA Surveillance: Molecular Approaches in Transcript Quality Control and their Implications in Clinical Diseases

PubMed Central

Moraes, Karen CM

2010-01-01

Production of mature mRNAs that encode functional proteins involves highly complex pathways of synthesis, processing and surveillance. At numerous steps during the maturation process, the mRNA transcript undergoes scrutiny by cellular quality control machinery. This extensive RNA surveillance ensures that only correctly processed mature mRNAs are translated and precludes production of aberrant transcripts that could encode mutant or possibly deleterious proteins. Recent advances in elucidating the molecular mechanisms of mRNA processing have demonstrated the existence of an integrated network of events, and have revealed that a variety of human diseases are caused by disturbances in the well-coordinated molecular equilibrium of these events. From a medical perspective, both loss and gain of function are relevant, and a considerable number of different diseases exemplify the importance of the mechanistic function of RNA surveillance in a cell. Here, mechanistic hallmarks of mRNA processing steps are reviewed, highlighting the medical relevance of their deregulation and how the understanding of such mechanisms can contribute to the development of therapeutic strategies. PMID:19829759

The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins

PubMed Central

Hessle, Viktoria; Björk, Petra; Sokolowski, Marcus; de Valdivia, Ernesto González; Silverstein, Rebecca; Artemenko, Konstantin; Tyagi, Anu; Maddalo, Gianluca; Ilag, Leopold; Helbig, Roger; Zubarev, Roman A.

2009-01-01

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins. PMID:19494042

tRNA biology charges to the front

PubMed Central

Phizicky, Eric M.; Hopper, Anita K.

2010-01-01

tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis. PMID:20810645

Comparative studies of two methods for miRNA isolation from milk whey.

PubMed

Jin, Xiao-lu; Wei, Zi-hai; Liu, Lan; Liu, Hong-yun; Liu, Jian-xin

2015-06-01

MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS(®) followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS(®) followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (<200 nt) from milk whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100).

Comparative studies of two methods for miRNA isolation from milk whey*

PubMed Central

Jin, Xiao-lu; Wei, Zi-hai; Liu, Lan; Liu, Hong-yun; Liu, Jian-xin

2015-01-01

MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS® followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS® followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (<200 nt) from milk whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100). PMID:26055915

Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

PubMed

Johansson, Marcus J O

2017-01-01

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity.

PubMed

Rialdi, Alexander; Hultquist, Judd; Jimenez-Morales, David; Peralta, Zuleyma; Campisi, Laura; Fenouil, Romain; Moshkina, Natasha; Wang, Zhen Zhen; Laffleur, Brice; Kaake, Robyn M; McGregor, Michael J; Haas, Kelsey; Pefanis, Evangelos; Albrecht, Randy A; Pache, Lars; Chanda, Sumit; Jen, Joanna; Ochando, Jordi; Byun, Minji; Basu, Uttiya; García-Sastre, Adolfo; Krogan, Nevan; van Bakel, Harm; Marazzi, Ivan

2017-05-04

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

PubMed

Román, Belén; González-Verdejo, Clara I; Peña, Francisco; Nadal, Salvador; Gómez, Pedro

2012-01-01

Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue. Copyright © 2012 John Wiley & Sons, Ltd.

External Quality Assessment for Rubella Virus RNA Detection Using Armored RNA in China.

PubMed

Zhang, D; Lin, G; Yi, L; Hao, M; Fan, G; Yang, X; Peng, R; Ding, J; Zhang, K; Zhang, R; Li, J

2017-02-01

Although tremendous efforts have been made to reduce rubella incidence, there are still 300 new cases of congenital rubella syndrome daily; thus, rubella infections remain one of the leading causes of preventable congenital birth defects. An effective surveillance system, which could be achieved and maintained by using an external quality assessment program, is critical for prevention and control of this disease. Armored RNAs, which are noninfectious and RNase-resistant, were used for encapsulation of the E1 gene of rubella virus and for preparation of a 10-specimen panel for external quality assessment. Thirty-two laboratories across mainland China that used nucleic acid tests for rubella virus RNA detection were included in the external quality assessment program organized by the National Center for Clinical Laboratories of China. Different kinds of commercial kits were used by the laboratories for nucleic acid extraction and TaqMan real-time reverse-transcription PCR for rubella virus RNA detection; 99.2% sensitivity and 100% specificity were achieved in this external quality assessment program. Most of the participating laboratories obtained accurate results for rubella nucleic acid tests, thereby achieving the quality required for regional rubella and congenital rubella syndrome elimination.

Exploring Fusarium head blight disease control by RNA interference

USDA-ARS?s Scientific Manuscript database

RNA interference (RNAi) technology provides a novel tool to study gene function and plant protection strategies. Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and quality by producing trichothecene mycotoxins including 3-acetyl deoxynivalenol (3-ADO...

Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

PubMed

Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

2017-10-06

Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

Protecting the proteome: Eukaryotic cotranslational quality control pathways

PubMed Central

2014-01-01

The correct decoding of messenger RNAs (mRNAs) into proteins is an essential cellular task. The translational process is monitored by several quality control (QC) mechanisms that recognize defective translation complexes in which ribosomes are stalled on substrate mRNAs. Stalled translation complexes occur when defects in the mRNA template, the translation machinery, or the nascent polypeptide arrest the ribosome during translation elongation or termination. These QC events promote the disassembly of the stalled translation complex and the recycling and/or degradation of the individual mRNA, ribosomal, and/or nascent polypeptide components, thereby clearing the cell of improper translation products and defective components of the translation machinery. PMID:24535822

Informatics for RNA Sequencing: A Web Resource for Analysis on the Cloud

PubMed Central

Griffith, Malachi; Walker, Jason R.; Spies, Nicholas C.; Ainscough, Benjamin J.; Griffith, Obi L.

2015-01-01

Massively parallel RNA sequencing (RNA-seq) has rapidly become the assay of choice for interrogating RNA transcript abundance and diversity. This article provides a detailed introduction to fundamental RNA-seq molecular biology and informatics concepts. We make available open-access RNA-seq tutorials that cover cloud computing, tool installation, relevant file formats, reference genomes, transcriptome annotations, quality-control strategies, expression, differential expression, and alternative splicing analysis methods. These tutorials and additional training resources are accompanied by complete analysis pipelines and test datasets made available without encumbrance at www.rnaseq.wiki. PMID:26248053

Evaluation of external RNA controls for the standardisation of gene expression biomarker measurements.

PubMed

Devonshire, Alison S; Elaswarapu, Ramnath; Foy, Carole A

2010-11-24

Gene expression profiling is an important approach for detecting diagnostic and prognostic biomarkers, and predicting drug safety. The development of a wide range of technologies and platforms for measuring mRNA expression makes the evaluation and standardization of transcriptomic data problematic due to differences in protocols, data processing and analysis methods. Thus, universal RNA standards, such as those developed by the External RNA Controls Consortium (ERCC), are proposed to aid validation of research findings from diverse platforms such as microarrays and RT-qPCR, and play a role in quality control (QC) processes as transcriptomic profiling becomes more commonplace in the clinical setting. Panels of ERCC RNA standards were constructed in order to test the utility of these reference materials (RMs) for performance characterization of two selected gene expression platforms, and for discrimination of biomarker profiles between groups. The linear range, limits of detection and reproducibility of microarray and RT-qPCR measurements were evaluated using panels of RNA standards. Transcripts of low abundance (≤ 10 copies/ng total RNA) showed more than double the technical variability compared to higher copy number transcripts on both platforms. Microarray profiling of two simulated 'normal' and 'disease' panels, each consisting of eight different RNA standards, yielded robust discrimination between the panels and between standards with varying fold change ratios, showing no systematic effects due to different labelling and hybridization runs. Also, comparison of microarray and RT-qPCR data for fold changes showed agreement for the two platforms. ERCC RNA standards provide a generic means of evaluating different aspects of platform performance, and can provide information on the technical variation associated with quantification of biomarkers expressed at different levels of physiological abundance. Distinct panels of standards serve as an ideal quality control tool kit for determining the accuracy of fold change cut-off threshold and the impact of experimentally-derived noise on the discrimination of normal and disease profiles.

Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

PubMed Central

Pichler, Martin; Zatloukal, Kurt

2013-01-01

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples. PMID:23936242

RNA-seq mixology: designing realistic control experiments to compare protocols and analysis methods

PubMed Central

Holik, Aliaksei Z.; Law, Charity W.; Liu, Ruijie; Wang, Zeya; Wang, Wenyi; Ahn, Jaeil; Asselin-Labat, Marie-Liesse; Smyth, Gordon K.

2017-01-01

Abstract Carefully designed control experiments provide a gold standard for benchmarking different genomics research tools. A shortcoming of many gene expression control studies is that replication involves profiling the same reference RNA sample multiple times. This leads to low, pure technical noise that is atypical of regular studies. To achieve a more realistic noise structure, we generated a RNA-sequencing mixture experiment using two cell lines of the same cancer type. Variability was added by extracting RNA from independent cell cultures and degrading particular samples. The systematic gene expression changes induced by this design allowed benchmarking of different library preparation kits (standard poly-A versus total RNA with Ribozero depletion) and analysis pipelines. Data generated using the total RNA kit had more signal for introns and various RNA classes (ncRNA, snRNA, snoRNA) and less variability after degradation. For differential expression analysis, voom with quality weights marginally outperformed other popular methods, while for differential splicing, DEXSeq was simultaneously the most sensitive and the most inconsistent method. For sample deconvolution analysis, DeMix outperformed IsoPure convincingly. Our RNA-sequencing data set provides a valuable resource for benchmarking different protocols and data pre-processing workflows. The extra noise mimics routine lab experiments more closely, ensuring any conclusions are widely applicable. PMID:27899618

Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination.

PubMed

Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

2016-01-01

Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with appropriate UEV collection procedure. We also suggested that a minimum 250 ml of urine should be processed to gather enough RNA for robust quantification, qualification and downstream analysis.

Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

PubMed Central

Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

2016-01-01

Background Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with appropriate UEV collection procedure. We also suggested that a minimum 250 ml of urine should be processed to gather enough RNA for robust quantification, qualification and downstream analysis. PMID:27345058

Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors.

PubMed

Pop, Laura A; Pileczki, Valentina; Cojocneanu-Petric, Roxana M; Petrut, Bogdan; Braicu, Cornelia; Jurj, Ancuta M; Buiga, Rares; Achimas-Cadariu, Patriciu; Berindan-Neagoe, Ioana

2016-01-01

Sample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies. The study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers. Every step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA. Gene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens.

Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors

PubMed Central

Pop, Laura A; Pileczki, Valentina; Cojocneanu-Petric, Roxana M; Petrut, Bogdan; Braicu, Cornelia; Jurj, Ancuta M; Buiga, Rares; Achimas-Cadariu, Patriciu; Berindan-Neagoe, Ioana

2016-01-01

Background Sample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies. Patients and methods The study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers. Results Every step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA. Conclusion Gene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens. PMID:27330317

A universal reference sample derived from clone vector for improved detection of differential gene expression

PubMed Central

Khan, Rishi L; Gonye, Gregory E; Gao, Guang; Schwaber, James S

2006-01-01

Background Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs™. Results We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. Conclusion Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes. PMID:16677381

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Lai, S.; Klotz, R.; Popova, Y.; Chakravarty, K.; Beegle, J. E.; Wigley, C. L.; Globus, R. K.

2014-01-01

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development. Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science return. Livers and spleens from mice were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection procedures including storage at minus 80 degrees Centigrade for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RNA Integrity Number (RIN) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNA, later) and liver (fast-freezing) at various time points post-euthanasia (from 5 minutes up to 105 minutes). The RNA recovered was of high quality (spleen, RIN greater than 8; liver, RIN greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at 105 minutes. Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) High-quality RNA samples from many different tissues can be recovered by dissection following prolonged storage of the tissue in situ at minus 80 degrees Centigrade. These findings have relevance both to high-value, ground-based experiments when sample collection capability is severely constrained, and to future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control.

PubMed

Kesanakurti, Prasad; Belton, Mark; Saeed, Hanaa; Rast, Heidi; Boyes, Ian; Rott, Michael

2016-10-01

The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS). Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

An exploratory analysis of the effects of a weight loss plus exercise program on cellular quality control mechanisms in older overweight women.

PubMed

Wohlgemuth, Stephanie E; Lees, Hazel A; Marzetti, Emanuele; Manini, Todd M; Aranda, Juan M; Daniels, Michael J; Pahor, Marco; Perri, Michael G; Leeuwenburgh, Christian; Anton, Stephen D

2011-06-01

Obese older adults are particularly susceptible to sarcopenia and have a higher prevalence of disability than their peers of normal weight. Interventions to improve body composition in late life are crucial to maintaining independence. The main mechanisms underlying sarcopenia have not been determined conclusively, but chronic inflammation, apoptosis, and impaired mitochondrial function are believed to play important roles. It has yet to be determined whether impaired cellular quality control mechanisms contribute to this process. The objective of this study was to assess the effects of a 6-month weight loss program combined with moderate-intensity exercise on the cellular quality control mechanisms autophagy and ubiquitin-proteasome, as well as on inflammation, apoptosis, and mitochondrial function, in the skeletal muscle of older obese women. The intervention resulted in significant weight loss (8.0 ± 3.9 % vs. 0.4 ± 3.1% of baseline weight, p = 0.002) and improvements in walking speed (reduced time to walk 400 meters, - 20.4 ± 16% vs. - 2.5 ± 12%, p = 0.03). In the intervention group, we observed a three-fold increase in messenger RNA (mRNA) levels of the autophagy regulators LC3B, Atg7, and lysosome-associated membrane protein-2 (LAMP-2) compared to controls. Changes in mRNA levels of FoxO3A and its targets MuRF1, MAFBx, and BNIP3 were on average seven-fold higher in the intervention group compared to controls, but these differences were not statistically significant. Tumor necrosis factor-α (TNF-α) mRNA levels were elevated after the intervention, but we did not detect significant changes in the downstream apoptosis markers caspase 8 and 3. Mitochondrial biogenesis markers (PGC1α and TFAm) were increased by the intervention, but this was not accompanied by significant changes in mitochondrial complex content and activity. In conclusion, although exploratory in nature, this study is among the first to report the stimulation of cellular quality control mechanisms elicited by a weight loss and exercise program in older obese women.

Up-Regulation of Autophagy in Small Intestine Paneth Cells in Response to Total-Body gamma-Irradiation

DTIC Science & Technology

2009-01-01

standard error of the mean (SEM). Analysis of variance procedures with Tukey post hoc correction examined the existence and nature of temporal trends ...apoptosis. Cell 2006;126:121–134. 20. Yorimitsu T, Klionsky DJ. Eating the enoplasmic reticulum: quality control by autophagy. Trends Cell Biol 2007;17...oxide signaling to iron- regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

PubMed

de Cremoux, P; Bieche, I; Tran-Perennou, C; Vignaud, S; Boudou, E; Asselain, B; Lidereau, R; Magdelénat, H; Becette, V; Sigal-Zafrani, B; Spyratos, F

2004-09-01

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

Use of External Quality Control Material for HIV-1 RNA Testing To Assess the Comparability of Data Generated in Separate Laboratories and the Stability of HIV-1 RNA in Samples after Prolonged Storage.

PubMed

Jennings, Cheryl; Wager, Carrie G; Scianna, Salvatore R; Zaccaro, Daniel J; Couzens, Amy; Mellors, John W; Coombs, Robert W; Bremer, James W

2018-06-01

The National Institute of Allergy and Infectious Diseases (NIAID) AIDS Clinical Trials Group (ACTG) stores specimens from its clinical trials in a biorepository and permits the use of these specimens for nonprotocol exploratory studies, once the studies for the original protocol are concluded. We sought to assess the comparability of the data generated from real-time HIV-1 RNA testing during two clinical trials with the data generated from the retesting of different aliquots of the same samples after years of storage at -80°C. Overall, there was 92% agreement in the data generated for 1,570 paired samples (kappa statistic = 0.757; 95% confidence interval [CI], 0.716 to 0.797), where samples were tested in one laboratory using the microwell plate (MWP) version of the Roche HIV-1 Monitor test within 1 to 37 days of collection and retested in another laboratory using the Cobas version of the assay after a median of 6.7 years of storage (range, 5.7 to 8.6 years). Historical external quality control data submitted to the NIAID Virology Quality Assurance program (VQA) by client laboratories using the same two versions of the Monitor assay were used to differentiate between systematic differences in the assays to evaluate the stability of HIV-1 RNA in the stored samples. No significant loss of RNA was noted in samples containing either a low concentration (<50 copies/ml) or a high concentration (≥50 copies/ml) of HIV-1 RNA ( P = 0.10 and P = 0.90, respectively) regardless of the time in storage. These data confirm the quality of the plasma samples in the ACTG biorepository following long-term storage. Copyright © 2018 American Society for Microbiology.

Fit for purpose frozen tissue collections by RNA integrity number-based quality control assurance at the Erasmus MC tissue bank.

PubMed

Kap, Marcel; Oomen, Monique; Arshad, Shazia; de Jong, Bas; Riegman, Peter

2014-04-01

About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80°C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN ≥6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN <5: not reliable for demanding downstream analysis; 5 ≤RIN <6: suitable for RT-qPCR; 6 ≤RIN <8: suitable for gene array analysis; and RIN ≥8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN ≥6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made.

RNA-seq Data: Challenges in and Recommendations for Experimental Design and Analysis.

PubMed

Williams, Alexander G; Thomas, Sean; Wyman, Stacia K; Holloway, Alisha K

2014-10-01

RNA-seq is widely used to determine differential expression of genes or transcripts as well as identify novel transcripts, identify allele-specific expression, and precisely measure translation of transcripts. Thoughtful experimental design and choice of analysis tools are critical to ensure high-quality data and interpretable results. Important considerations for experimental design include number of replicates, whether to collect paired-end or single-end reads, sequence length, and sequencing depth. Common analysis steps in all RNA-seq experiments include quality control, read alignment, assigning reads to genes or transcripts, and estimating gene or transcript abundance. Our aims are two-fold: to make recommendations for common components of experimental design and assess tool capabilities for each of these steps. We also test tools designed to detect differential expression, since this is the most widespread application of RNA-seq. We hope that these analyses will help guide those who are new to RNA-seq and will generate discussion about remaining needs for tool improvement and development. Copyright © 2014 John Wiley & Sons, Inc.

Transfer RNA: a dancer between charging and mis-charging for protein biosynthesis.

PubMed

Zhou, Xiaolong; Wang, Enduo

2013-10-01

Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the protein. To perform its role in protein biosynthesis, it has to be accurately recognized by aminoacyl-tRNA synthetases (aaRSs) to generate aminoacyl-tRNAs (aa-tRNAs). The correct pairing between an amino acid with its cognate tRNA is crucial for translational quality control. Production and utilization of mis-charged tRNAs are usually detrimental for all the species, resulting in cellular dysfunctions. Correct aa-tRNAs formation is collectively controlled by aaRSs with distinct mechanisms and/or other trans-factors. However, in very limited instances, mis-charged tRNAs are intermediate for specific pathways or essential components for the translational machinery. Here, from the point of accuracy in tRNA charging, we review our understanding about the mechanism ensuring correct aa-tRNA generation. In addition, some unique mis-charged tRNA species necessary for the organism are also briefly described.

Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore

PubMed Central

Hessle, Viktoria; von Euler, Anne; González de Valdivia, Ernesto; Visa, Neus

2012-01-01

Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs. PMID:22745224

A protocol for preparing, characterizing and using three RNA-specific, live cell imaging probes: E36, E144 and F22.

PubMed

Li, Qian; Chang, Young-Tae

2006-01-01

This protocol outlines a methodology for the preparation and characterization of three RNA-specific fluorescent probes (E36, E144 and F22) and their use in live cell imaging. It describes a detailed procedure for their chemical synthesis and purification; serial product characterization and quality control tests, including measurements of their fluorescence properties in solution, measurement of RNA specificity and analysis of cellular toxicity; and live cell staining and counterstaining with Hoechst or DAPI. Preparation and application of these RNA imaging probes takes 1 week.

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

PubMed Central

Soheilypour, M.; Mofrad, M. R. K.

2016-01-01

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

PubMed

Soheilypour, M; Mofrad, M R K

2016-11-02

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

PubMed

Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

2017-07-07

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNA Lys (UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Novel small interfering RNA-containing solution protecting donor organs in heart transplantation.

PubMed

Zheng, Xiufen; Lian, Dameng; Wong, Arthur; Bygrave, Michael; Ichim, Thomas E; Khoshniat, Mahdieh; Zhang, Xusheng; Sun, Hongtao; De Zordo, Tobias; Lacefield, James C; Garcia, Bertha; Jevnikar, Anthony M; Min, Wei-Ping

2009-09-22

Ischemia/reperfusion injury is a major factor in graft quality and subsequent function in the transplantation setting. We hypothesize that the process of RNA interference may be used to "engineer" a graft to suppress expression of genes associated with inflammation, apoptosis, and complement, which are believed to cause ischemia/reperfusion injury. Such manipulation of pathological gene expression may be performed by treatment of the graft ex vivo with small interfering RNA (siRNA) as part of the preservation procedure. Heart grafts from BALB/c mice were preserved in UW solution (control) or UW solution containing siRNAs targeting tumor necrosis factor-alpha, C3, and Fas genes (siRNA solution) at 4 degrees C for 48 hours and subsequently transplanted into syngeneic recipients. Tumor necrosis factor-alpha, C3, and Fas genes were elevated by ischemia/reperfusion injury after 48 hours of preservation in UW solution. Preservation in siRNA solution knocked down gene expression at the level of messenger RNA and protein in the grafts after transplantation. All grafts preserved in siRNA solution showed strong contraction, whereas grafts preserved in control solution demonstrated no detectable contraction by high-frequency ultrasound scanning. siRNA solution-treated organs exhibited improved histology and diminished neutrophil and lymphocyte infiltration compared with control solution-treated organs. Furthermore, the treated heart grafts retained strong beating up to the end of the observation period (>100 days), whereas all control grafts lost function within 8 days. Incorporation of siRNA into organ storage solution is a feasible and effective method of attenuating ischemia/reperfusion injury, protecting cardiac function, and prolonging graft survival.

Clinical implementation of RNA signatures for pharmacogenomic decision-making

PubMed Central

Tang, Weihua; Hu, Zhiyuan; Muallem, Hind; Gulley, Margaret L

2011-01-01

RNA profiling is increasingly used to predict drug response, dose, or toxicity based on analysis of drug pharmacokinetic or pharmacodynamic pathways. Before implementing multiplexed RNA arrays in clinical practice, validation studies are carried out to demonstrate sufficient evidence of analytic and clinical performance, and to establish an assay protocol with quality assurance measures. Pathologists assure quality by selecting input tissue and by interpreting results in the context of the input tissue as well as the technologies that were used and the clinical setting in which the test was ordered. A strength of RNA profiling is the array-based measurement of tens to thousands of RNAs at once, including redundant tests for critical analytes or pathways to promote confidence in test results. Instrument and reagent manufacturers are crucial for supplying reliable components of the test system. Strategies for quality assurance include careful attention to RNA preservation and quality checks at pertinent steps in the assay protocol, beginning with specimen collection and proceeding through the various phases of transport, processing, storage, analysis, interpretation, and reporting. Specimen quality is checked by probing housekeeping transcripts, while spiked and exogenous controls serve as a check on analytic performance of the test system. Software is required to manipulate abundant array data and present it for interpretation by a laboratory physician who reports results in a manner facilitating therapeutic decision-making. Maintenance of the assay requires periodic documentation of personnel competency and laboratory proficiency. These strategies are shepherding genomic arrays into clinical settings to provide added value to patients and to the larger health care system. PMID:23226056

Rarely at rest

PubMed Central

Hardwick, Steven W.; Luisi, Ben F.

2013-01-01

RNA helicases are compact, machine-like proteins that can harness the energy of nucleoside triphosphate binding and hydrolysis to dynamically remodel RNA structures and protein-RNA complexes. Through such activities, helicases participate in virtually every process associated with the expression of genetic information. Often found as components of multi-enzyme assemblies, RNA helicases facilitate the processivity of RNA degradation, the remodeling of protein interactions during maturation of structured RNA precursors, and fidelity checks of RNA quality. In turn, the assemblies modulate and guide the activities of the helicases. We describe the roles of RNA helicases with a conserved “DExD/H box” sequence motif in representative examples of such machineries from bacteria, archaea and eukaryotes. The recurrent occurrence of such helicases in complex assemblies throughout the course of evolution suggests a common requirement for their activities to meet cellular demands for the dynamic control of RNA metabolism. PMID:23064154

Origins of tmRNA: the missing link in the birth of protein synthesis?

PubMed

Macé, Kevin; Gillet, Reynald

2016-09-30

The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The FDA's Experience with Emerging Genomics Technologies-Past, Present, and Future.

PubMed

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-07-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing.

The FDA’s Experience with Emerging Genomics Technologies—Past, Present, and Future

PubMed Central

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-01-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing. PMID:27116022

Chronic Heat Stress Impairs the Quality of Breast-Muscle Meat in Broilers by Affecting Redox Status and Energy-Substance Metabolism.

PubMed

Lu, Zhuang; He, Xiaofang; Ma, Bingbing; Zhang, Lin; Li, Jiaolong; Jiang, Yun; Zhou, Guanghong; Gao, Feng

2017-12-27

We investigated the molecular mechanisms by which chronic heat stress impairs the breast-meat quality of broilers. Broilers were assigned to three groups: the normal control (NC) group, heat-stress (HS) group, and pair-fed (PF) group. After 7 days of heat exposure (32 °C), the high temperature had caused oxidative stress; elevated the activity of citrate synthase (CS), the mRNA expression of M-CPT1, and the phosphorylation level of AMPKα; and reduced the mRNA expression of avUCP. After 14 days of heat exposure, the heat stress had increased the lightness and drip loss and decreased the pH and shear force of the breast meat. Additionally, the heat exposure had increased the mRNA expressions of FAS, ACC, and PDK4; the content of lipids; and the activities of lactic dehydrogenase and pyruvate kinase, and it had decreased the mRNA expression of M-CPT1 and the activity of CS. In conclusion, chronic heat stress impairs meat quality by causing mitochondria to malfunction and affecting energy-substance aerobic metabolism, resulting in increased glycolysis and intramuscular fat deposition.

Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation.

PubMed

Eikmans, Michael; Rekers, Niels V; Anholts, Jacqueline D H; Heidt, Sebastiaan; Claas, Frans H J

2013-03-14

Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.

Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow

PubMed Central

Buschmann, Dominik; Haberberger, Anna; Kirchner, Benedikt; Spornraft, Melanie; Riedmaier, Irmgard; Schelling, Gustav; Pfaffl, Michael W.

2016-01-01

Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will encourage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions. PMID:27317696

Comparison of RNA Isolation Methods From Insect Larvae

PubMed Central

Ridgeway, J. A.; Timm, A. E.

2014-01-01

Abstract Isolating RNA from insects is becoming increasingly important in molecular entomology. Four methods including three commercial kits RNeasy Mini Kit (Qiagen), SV Total RNA isolation system (Promega), TRIzol reagent (Invitrogen), and a cetyl trimethylammonium bromide (CTAB)-based method were compared regarding their ability to isolate RNA from whole-body larvae of Thaumatotibia leucotreta (Meyrick), Thanatophilus micans (F.), Plutella xylostella (L.), and Tenebrio molitor (L.). A difference was observed among the four methods regarding RNA quality but not quantity. However, RNA quality and quantity obtained was not dependent on the insect species. The CTAB-based method produced low-quality RNA and the Trizol reagent produced partially degraded RNA, whereas the RNeasy Mini Kit and SV Total RNA isolation system produced RNA of consistently high quality. However, after reverse transcription to cDNA, RNA produced using all four extraction methods could be used to successfully amplify a 708 bp fragment of the cytochrome oxidase I gene. Of the four methods, the SV Total RNA isolation system showed the least amount of DNA contamination with the highest RNA integrity number and is thus recommended for stringent applications where high-quality RNA is required. This is the first comparison of RNA isolation methods among different insect species and the first to compare RNA isolation methods in insects in the last 20 years. PMID:25527580

Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for next-generation sequencing.

PubMed

Pérez-Portela, Rocío; Riesgo, Ana

2013-09-01

Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high-quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash-frozen and stored at -80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at -80 °C, RNAlater and storage at -20 °C, preservation in TRIzol reagent and storage at -80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips. © 2013 John Wiley & Sons Ltd.

Isolation of high-quality total RNA from leaves of Myrciaria dubia "CAMU CAMU".

PubMed

Gómez, Juan Carlos Castro; Reátegui, Alina Del Carmen Egoavil; Flores, Julián Torres; Saavedra, Roberson Ramírez; Ruiz, Marianela Cobos; Correa, Sixto Alfredo Imán

2013-01-01

Myrciaria dubia is a main source of vitamin C for people in the Amazon region. Molecular studies of M. dubia require high-quality total RNA from different tissues. So far, no protocols have been reported for total RNA isolation from leaves of this species. The objective of this research was to develop protocols for extracting high-quality total RNA from leaves of M. dubia. Total RNA was purified following two modified protocols developed for leaves of other species (by Zeng and Yang, and by Reid et al.) and one modified protocol developed for fruits of the studied species (by Silva). Quantity and quality of purified total RNA were assessed by spectrophotometric and electrophoretic analysis. Additionally, quality of total RNA was evaluated with reverse-transcription polymerase chain reaction (RT-PCR). With these three modified protocols we were able to isolate high-quality RNA (A260nm/A280nm >1.9 and A260nm/A230nm >2.0). Highest yield was produced with the Zeng and Yang modified protocol (384±46µg ARN/g fresh weight). Furthermore, electrophoretic analysis showed the integrity of isolated RNA and the absence of DNA. Another proof of the high quality of our purified RNA was the successful cDNA synthesis and amplification of a segment of the M. dubia actin 1 gene. We report three modified protocols for isolation total RNA from leaves of M. dubia. The modified protocols are easy, rapid, low in cost, and effective for high-quality and quantity total RNA isolation suitable for cDNA synthesis and polymerase chain reaction.

Preservation of Fine-Needle Aspiration Specimens for Future Use in RNA-Based Molecular Testing

PubMed Central

Ladd, Amy C.; O'Sullivan-Mejia, Emerald; Lea, Tasha; Perry, Jessica; Dumur, Catherine I.; Dragoescu, Ema; Garrett, Carleton T.; Powers, Celeste N.

2015-01-01

Background 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. Methods 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). Results 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. Conclusions 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. PMID:21287691

A technical assessment of the porcine ejaculated spermatozoa for a sperm-specific RNA-seq analysis.

PubMed

Gòdia, Marta; Mayer, Fabiana Quoos; Nafissi, Julieta; Castelló, Anna; Rodríguez-Gil, Joan Enric; Sánchez, Armand; Clop, Alex

2018-04-26

The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPure TM ; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome. FPKM: fragments per kilobase of transcript per million mapped reads; KRT1: keratin 1; miRNA: micro-RNA; miscRNA: miscellaneous RNA; Mt rRNA: mitochondrial ribosomal RNA; Mt tRNA: mitochondrial transference RNA; OAZ3: ornithine decarboxylase antizyme 3; ORT: osmotic resistance test; piRNA: Piwi-interacting RNA; PRM1: protamine 1; PTPRC: protein tyrosine phosphatase receptor type C; rRNA: ribosomal RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRR: sperm recovery rate; tRNA: transfer RNA.

A robust method for RNA extraction and purification from a single adult mouse tendon.

PubMed

Grinstein, Mor; Dingwall, Heather L; Shah, Rishita R; Capellini, Terence D; Galloway, Jenna L

2018-01-01

Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts. Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed. After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons. Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.

RNA-Seq reveals MicroRNA expression signature and genetic polymorphism associated with growth and muscle quality traits in rainbow trout

USDA-ARS?s Scientific Manuscript database

The role of microRNA expression and genetic variation in microRNA-binding sites of target genes on growth and muscle quality traits is poorly characterized. We used RNA-Seq approach to investigate their importance on 5 growth and muscle quality traits: whole body weight (WBW), muscle yield, muscle c...

Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization.

PubMed

Forsgren, Eva; Locke, Barbara; Semberg, Emilia; Laugen, Ane T; Miranda, Joachim R de

2017-08-01

Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling-transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at -80°C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder™ homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15min. Copyright © 2017 Elsevier B.V. All rights reserved.

The nuclear tRNA aminoacylation-dependent pathway may be the principal route used to export tRNA from the nucleus in Saccharomyces cerevisiae.

PubMed

Steiner-Mosonyi, Marta; Mangroo, Dev

2004-03-15

Nuclear tRNA export in Saccharomyces cerevisiae has been proposed to involve three pathways, designated Los1p-dependent, Los1p-independent nuclear aminoacylation-dependent, and Los1p- and nuclear aminoacylation-independent. Here, a comprehensive biochemical analysis was performed to identify tRNAs exported by the aminoacylation-dependent and -independent pathways of S. cerevisiae. Interestingly, the major tRNA species of at least 19 families were found in the aminoacylated form in the nucleus. tRNAs known to be exported by the export receptor Los1p were also aminoacylated in the nucleus of both wild-type and mutant Los1p strains. FISH (fluorescence in situ hybridization) analyses showed that tRNA(Tyr) co-localizes with the U18 small nucleolar RNA in the nucleolus of a tyrosyl-tRNA synthetase mutant strain defective in nuclear tRNA(Tyr) export because of a block in nuclear tRNA(Tyr) aminoacylation. tRNA(Tyr) was also found in the nucleolus of a utp8 mutant strain defective in nuclear tRNA export but not nuclear tRNA aminoacylation. These results strongly suggest that the nuclear aminoacylation-dependent pathway is principally responsible for tRNA export in S. cerevisiae and that Los1p is an export receptor of this pathway. It is also likely that in mammalian cells tRNAs are mainly exported from the nucleus by the nuclear aminoacylation-dependent pathway. In addition, the data are consistent with the idea that nuclear aminoacylation is used as a quality control mechanism for ensuring nuclear export of only mature and functional tRNAs, and that this quality assurance step occurs in the nucleolus.

Evaluating Quality of Aged Archival Formalin-Fixed Paraffin-Embedded Samples for RNA-Sequencing

EPA Science Inventory

Archival formalin-fixed paraffin-embedded (FFPE) samples offer a vast, untapped source of genomic data for biomarker discovery. However, the quality of FFPE samples is often highly variable, and conventional methods to assess RNA quality for RNA-sequencing (RNA-seq) are not infor...

RNase-Resistant Virus-Like Particles Containing Long Chimeric RNA Sequences Produced by Two-Plasmid Coexpression System▿

PubMed Central

Wei, Yuxiang; Yang, Changmei; Wei, Baojun; Huang, Jie; Wang, Lunan; Meng, Shuang; Zhang, Rui; Li, Jinming

2008-01-01

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments—hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)—was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 106 to 102 copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection. PMID:18305135

Role of Protein Quality Control Failure in Alcoholic Hepatitis Pathogenesis.

PubMed

French, Samuel W; Masouminia, Maryam; Samadzadeh, Sara; Tillman, Brittany C; Mendoza, Alejandro; French, Barbara A

2017-02-08

The mechanisms of protein quality control in hepatocytes in cases of alcoholic hepatitis (AH) including ufmylation, FAT10ylation, metacaspase 1 (Mca1), ERAD (endoplasmic reticulum-associated degradation), JUNQ (juxta nuclear quality control), IPOD (insoluble protein deposit) autophagocytosis, and ER stress are reviewed. The Mallory-Denk body (MDB) formation develops in the hepatocytes in alcoholic hepatitis as a consequence of the failure of these protein quality control mechanisms to remove misfolded and damaged proteins and to prevent MDB aggresome formation within the cytoplasm of hepatocytes. The proteins involved in the quality control pathways are identified, quantitated, and visualized by immunofluorescent antibody staining of liver biopsies from patients with AH. Quantification of the proteins are achieved by measuring the fluorescent intensity using a morphometric system. Ufmylation and FAT10ylation pathways were downregulated, Mca1 pathways were upregulated, autophagocytosis was upregulated, and ER stress PERK (protein kinase RNA-like endoplasmic reticulum kinase) and CHOP (CCAAT/enhancer-binding protein homologous protein) mechanisms were upregulated. Despite the upregulation of several pathways of protein quality control, aggresomes (MDBs) still formed in the hepatocytes in AH. The pathogenesis of AH is due to the failure of protein quality control, which causes balloon-cell change with MDB formation and ER stress.

Determining RNA quality for NextGen sequencing: some exceptions to the gold standard rule of 23S to 16S rRNA ratio

USDA-ARS?s Scientific Manuscript database

Using next-generation-sequencing technology to assess entire transcriptomes requires high quality starting RNA. Currently, RNA quality is routinely judged using automated microfluidic gel electrophoresis platforms and associated algorithms. Here we report that such automated methods generate false-n...

SARTools: A DESeq2- and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data.

PubMed

Varet, Hugo; Brillet-Guéguen, Loraine; Coppée, Jean-Yves; Dillies, Marie-Agnès

2016-01-01

Several R packages exist for the detection of differentially expressed genes from RNA-Seq data. The analysis process includes three main steps, namely normalization, dispersion estimation and test for differential expression. Quality control steps along this process are recommended but not mandatory, and failing to check the characteristics of the dataset may lead to spurious results. In addition, normalization methods and statistical models are not exchangeable across the packages without adequate transformations the users are often not aware of. Thus, dedicated analysis pipelines are needed to include systematic quality control steps and prevent errors from misusing the proposed methods. SARTools is an R pipeline for differential analysis of RNA-Seq count data. It can handle designs involving two or more conditions of a single biological factor with or without a blocking factor (such as a batch effect or a sample pairing). It is based on DESeq2 and edgeR and is composed of an R package and two R script templates (for DESeq2 and edgeR respectively). Tuning a small number of parameters and executing one of the R scripts, users have access to the full results of the analysis, including lists of differentially expressed genes and a HTML report that (i) displays diagnostic plots for quality control and model hypotheses checking and (ii) keeps track of the whole analysis process, parameter values and versions of the R packages used. SARTools provides systematic quality controls of the dataset as well as diagnostic plots that help to tune the model parameters. It gives access to the main parameters of DESeq2 and edgeR and prevents untrained users from misusing some functionalities of both packages. By keeping track of all the parameters of the analysis process it fits the requirements of reproducible research.

Optimizing RNA Extraction of Renal Papilla Biopsy Tissue in Kidney Stone Formers: A New Methodology for Genomic Study.

PubMed

Taguchi, Kazumi; Usawachintachit, Manint; Hamamoto, Shuzo; Unno, Rei; Tzou, David T; Sherer, Benjamin A; Wang, Yongmei; Okada, Atsushi; Stoller, Marshall L; Yasui, Takahiro; Chi, Thomas

2017-09-01

Endoscopic tools have provided versatile examination and treatment for kidney stone procedures. Despite endourologists researching urinary stone disease using endoscopes to collect tissue, this tissue collection method is limited. Endoscopically removed tissues are small in size, restricting the types of genome-based examination possible. We investigated a new method of renal papilla biopsy and RNA extraction to establish a genomic research methodology for kidney stone disease. We conducted a prospective multi-institutional study and collected renal papilla specimens from consecutive percutaneous nephrolithotomy and ureteroscopy (URS) cases performed for removal of upper urinary tract stones. Renal papilla tissue was extracted using ureteroscopic biopsy forceps after stone removal. RNA was extracted using two different extraction kits, and their quantity and quality were examined. Additionally, the impact of biopsy on surgical complications was compared between cases performed with and without biopsy by matched case-control analysis adjusted for age, gender, body mass index, bilaterality, and stone burden. A total of 90 biopsies from 49 patients were performed, and the median duration between specimen collection and RNA extraction was 61 days. Both univariate and multivariate analyses showed BIGopsy ® forceps usage significantly increased the total yield (p = 0.004) and quality (p = 0.001 for A260/280, p = 0.004 for A260/A230) of extracted RNA. Extraction using the RNeasy Micro Kit ® also improved A260/A230, whereas reduced RNA integrity number of extracted RNA by univariate and multivariate analyses (p = 0.002 and p < 0.001, respectively). Moreover, matched case-control study demonstrated that endoscopic renal papilla biopsy caused no significant surgical complications, including bleeding, decreased stone clearance and hematocrit, and renal dysfunction. Biopsies during URS imparted an average of 20 minutes of procedure time over nonbiopsy cases. We demonstrate a safe methodology for optimal RNA extraction of renal papilla tissue. This technique will accelerate advanced genomic studies for kidney stone formers by facilitating larger tissue yields.

A highly efficient method for extracting next-generation sequencing quality RNA from adipose tissue of recalcitrant animal species.

PubMed

Sharma, Davinder; Golla, Naresh; Singh, Dheer; Onteru, Suneel K

2018-03-01

The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species. © 2017 Wiley Periodicals, Inc.

Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus.

PubMed

Lin, Guigao; Zhang, Kuo; Zhang, Dong; Han, Yanxi; Xie, Jiehong; Li, Jinming

2017-03-01

The emergence of Zika virus demands accurate laboratory diagnostics. Nucleic acid testing is currently the definitive method for diagnosis of Zika infection. In 2016, an external quality assurance (EQA) for assessing the quality of molecular testing of Zika virus was carried out in China. A single armored RNA encapsulating a 4942-nucleotides (nt) long specific RNA sequence of Zika virus was prepared and used as positive samples. A pre-tested EQA panel, consisting of 4 negative and 6 positive samples with different concentrations of armored RNA, was distributed to 38 laboratories that perform molecular detection of Zika virus. A total of 39 data sets (1 laboratory used two test kits in parallel), produced by using commercial (n=38) or laboratory developed (n=1) quantitative reverse-transcriptase PCR (qRT-PCR) kits, were received. Of these, 35 (89.7%) had correct results for all 10 samples, and 4 (10.3%) reported at least 1 error (11 in total). The testing errors were all false-negatives, highlighting the need of improvements in detecting sensitivity. The EQA reveals that the majority of participating laboratories are proficient in molecular testing of Zika virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

PubMed Central

Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

2013-01-01

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing. PMID:24236095

Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank.

PubMed

Calleros-Basilio, Laura; Cortés, María Alicia; García-Jerez, Andrea; Luengo-Rodríguez, Alicia; Orozco-Agudo, Ana; Valdivielso, José Manuel; Rodríguez-Puyol, Diego; Rodríguez-Puyol, Manuel

2016-12-01

Biobanks are useful platforms to build bridges between basic, translational, and clinical research and clinical care. They are repositories of high-quality human biological samples ideal for evaluating their histological characteristics and also their genome, transcriptome, and proteome. The Spanish Renal Research Network Biobank contains more than 76,500 well-preserved frozen samples of a wide variety of kidney diseases, collected from 5450 patients seen by over 70 nephrology services throughout the Spanish territory. To determine and to report the results of the quality control of samples and processes conducted in our biobank, implemented in accordance with the requirements of the ISO 9001:2008 international standard. Two types of quality controls were performed: (1) systematic, that is, measurement of viable peripheral blood mononuclear cells (PBMCs) obtained and purity of nucleic acids and (2) ad-hoc, that is, viability of thawed PBMC, DNA extraction process reproducibility, and the integrity and functionality of nucleic acids, implemented on a routine basis. PBMC isolation by Ficoll yielded reproducible results and its cryopreserved viability was >90%. Acceptable A260/A280 ratios were obtained for the vast majority of the DNA (n = 2328) and RNA (n = 78) samples analyzed. DNA integrity was demonstrated by agarose gels and by β-globulin gene polymerase chain reaction (PCR) amplification of 1327 and 989 bp fragments. DNA of acceptable quality had at least three bands of β-globulin amplified obtained (n = 26/30). RNA integrity number (RIN) determinations obtained RIN numbers ≥7 (n = 87/96). The amplifiability of nucleic acids was confirmed by qPCR and RT-qPCR of β-actin and GAPDH genes. Long storage or delayed processing time did not affect the quality of the samples analyzed. The processes of DNA extraction also yielded reproducible results. These results clearly indicate that our PBMC, DNA, and RNA stored samples meet the required quality standards to be used for biomedical research, ensuring their long-term preservation.

A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.

PubMed

Haque, Ashraful; Engel, Jessica; Teichmann, Sarah A; Lönnberg, Tapio

2017-08-18

RNA sequencing (RNA-seq) is a genomic approach for the detection and quantitative analysis of messenger RNA molecules in a biological sample and is useful for studying cellular responses. RNA-seq has fueled much discovery and innovation in medicine over recent years. For practical reasons, the technique is usually conducted on samples comprising thousands to millions of cells. However, this has hindered direct assessment of the fundamental unit of biology-the cell. Since the first single-cell RNA-sequencing (scRNA-seq) study was published in 2009, many more have been conducted, mostly by specialist laboratories with unique skills in wet-lab single-cell genomics, bioinformatics, and computation. However, with the increasing commercial availability of scRNA-seq platforms, and the rapid ongoing maturation of bioinformatics approaches, a point has been reached where any biomedical researcher or clinician can use scRNA-seq to make exciting discoveries. In this review, we present a practical guide to help researchers design their first scRNA-seq studies, including introductory information on experimental hardware, protocol choice, quality control, data analysis and biological interpretation.

Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus

PubMed Central

Madi, Mikidache; Mioulet, Valerie; King, Donald P.; Lomonossoff, George P.; Montague, Nicholas P.

2015-01-01

Positive controls are an important component of the quality-control of molecular tests used for diagnosis of livestock diseases. For high consequence agents such as foot-and-mouth disease virus (FMDV), the positive controls required to monitor template extraction, reverse transcription and amplification steps usually consist of material derived from infectious viruses. Therefore, their production is dependent upon the use of high containment facilities and their deployment carries the risks associated with inactivation of “live” FMDV. This paper describes the development of a novel non-infectious positive control that encodes FMDV RNA sequences that are encapsidated within Cowpea mosaic virus (CPMV) particles. This surrogate RNA has been engineered to contain sequences from the 5′UTR and 3D regions of FMDV targeted by many molecular assays (conventional RT-PCR, real-time RT-PCR and RT-LAMP). These sequences were inserted into a movement-deficient version of CPMV RNA-2 which is rescued from cowpea plants (Vigna unguiculota) by inoculation with RNA-1. In order to evaluate the performance of these encapsidated RNAs, nucleic acid prepared from a 10-fold dilution series was tested using a range of molecular assays. Results generated by using the molecular assays confirmed RNA-dependent amplification and the suitability of these particles for use in a range of diagnostic tests. Moreover, these CPMV particles were highly stable for periods of up to 46 days at room temperature and 37 °C. Recombinant CPMV can be used to produce high yields of encapsidated RNAs that can be used as positive and negative controls and standards in molecular assays. This approach provides a surrogate that can be potentially used outside of containment laboratories as an alternative to inactivated infectious virus for molecular diagnostic testing. PMID:25864934

Bleach Gel: A Simple Agarose Gel for Analyzing RNA Quality

PubMed Central

Aranda, Patrick S.; LaJoie, Dollie M.; Jorcyk, Cheryl L.

2013-01-01

RNA-based applications requiring high quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the ‘bleach gel’ is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality. PMID:22222980

Human miRNome profiling in colorectal cancer and liver metastasis development

PubMed Central

Perilli, Lisa; Pizzini, Silvia; Bisognin, Andrea; Mandruzzato, Susanna; Biasiolo, Marta; Facciolli, Arianna; Rossi, Elisabetta; Esposito, Giovanni; Rugge, Massimo; Pilati, Pierluigi; Mocellin, Simone; Nitti, Donato; Bortoluzzi, Stefania; Zanovello, Paola

2014-01-01

Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colorectal cancer has mainly been demonstrated in primary tumors. The miRNA expression profiles in 78 samples from 46 patients were analyzed to identify changes in miRNA expression level among normal colon mucosa, primary tumor and liver metastasis samples. Using this dataset, we describe the interplay of miRNA groups in regulating pathways that are important for tumor development. Here we describe in details the contents and quality controls for the miRNA expression and clinical data associated with the study published by Pizzini and colleagues in the BMC Genomics in 2013 (Pizzini et al., 2013). Data are deposited in GEO database as GSE35834 series. PMID:26484092

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

PubMed Central

Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

2017-01-01

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC. PMID:28685749

A rapid and efficient SDS-based RNA isolation protocol from different tissues of coffee.

PubMed

Huded, Arun Kumar C; Jingade, Pavankumar; Mishra, Manoj Kumar

2018-03-01

Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6-454.8 µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 ( A 260/280 ) and 1.95 to 2.14 ( A 260/230 ), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65 °C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrome c oxidase gene-specific primers. The amplification of a single 392 bp fragment using cDNA and 1.5 kb fragment using genomic DNA samples confirmed the absence of DNA contamination. The present protocol is rapid and yielded good quality and quantity of RNA suitable for functional genomics studies.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions

PubMed Central

Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W.; Oegema, Karen; Desai, Arshad

2016-01-01

SUMMARY Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

A Multivariate Evaluation of Factors Affecting the Quality of Freshly Frozen Tissue Specimens.

PubMed

Wang, Tong-Hong; Chen, Chin-Chuan; Liang, Kung-Hao; Chen, Chi-Yuan; Chuang, Wen-Yu; Ueng, Shir-Hwa; Chu, Pao-Hsien; Huang, Chung-Guei; Chen, Tse-Ching; Hsueh, Chuen

2017-08-01

Well-prepared and preserved freshly frozen specimens are indispensable materials for clinical studies. To manage specimen quality and to understand the factors potentially affecting specimen quality during preservation processes, we analyzed the quality of RNA and genomic DNA of various tissues collected between 2002 and 2011 in Linkou Chang Gung Memorial Hospital, Taiwan. During this period, a total of 1059 freshly frozen specimens from eight major cancer categories were examined. It was found that preservation duration, organ origin, and tissue type could all influence the quality of RNA samples. The increased preservation period correlated with decreased RNA quality; the brain, breast, and stomach RNA specimens displayed faster degradation rates than those of other organs, and RNA specimens isolated from tumor tissues were apparently more stable than those of other tissues. These factors could all be used as quality predictors of RNA quality. In contrast, almost all analyses revealed that the genomic DNA samples had good quality, which was not influenced by the aforementioned factors. The results assisted us in determining preservation factors that affect specimen quality, which could provide evidence for improving processes of sample collection and preservation. Furthermore, the results are also useful for researchers to adopt as the evaluation criteria for choosing specimen collection and preservation strategies.

Identification of plasma microRNAs as a biomarker of sporadic Amyotrophic Lateral Sclerosis.

PubMed

Takahashi, Ikuko; Hama, Yuka; Matsushima, Masaaki; Hirotani, Makoto; Kano, Takahiro; Hohzen, Hideki; Yabe, Ichiro; Utsumi, Jun; Sasaki, Hidenao

2015-10-24

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, which leads to the loss of upper and lower motor neurons, with a currently unknown etiology. Specific biomarkers could help in early detection and diagnosis, and could also act as indicators of disease progression and therapy effectiveness. MicroRNAs (miRNAs) are small (18-25 nucleotides), single-stranded non-coding RNA molecules that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression, and are essential for nervous system development. Many of the genes associated with genetic ALS have pathological biological pathways related to RNA metabolism, and their pathogenesis may be affecting the maturing processes of miRNA. We compared miRNA from the plasma of sALS patients and healthy controls using two cohorts; a discovery cohort analyzed with microarray (16 sALS patients and ten healthy controls) and a validation cohort confirmed with qPCR (48 sALS patients, 47 healthy controls and 30 disease controls). We measured the total amount of extracted RNA along with a spike-in control that ensured the quality of our quantification. A percentage of the 10-40 nt RNAs extracted from the total RNA showed a significant increase in ALS patients. There was a negative correlation between total RNA concentration and disease duration from onset to end point. Three of the miRNAs were up-regulated and six were down-regulated significantly in the discovery cohort. Since an internal control is required as a sample stability indicator of both the patients and controls in microarray analysis, we selected the miRNA showing the smallest dispersion and equivalency between the two groups' mean value, and decided to use hsa-miR-4516. We found hsa-miR-4649-5p to be up-regulated, and hsa-miR-4299 to be down-regulated, where each was not influenced by clinical characteristics. EPHA4, a target gene linked to the nervous system which has also been reported to be a disease modifier of ALS, is the common and most notable target gene of hsa-miR-4649-5p and hsa-miR-4299. We have shown the relationship circulating plasma miRNA has with both healthy controls and diseased patients. Hsa-miR-4649-5p and hsa-miR-4299 have the potential to be ALS diagnosis biomarkers.

TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

Cancer.gov

Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

Method Optimization for Extracting High-Quality RNA From the Human Pancreas Tissue.

PubMed

Jun, Eunsung; Oh, Juyun; Lee, Song; Jun, Hye-Ryeong; Seo, Eun Hye; Jang, Jin-Young; Kim, Song Cheol

2018-06-01

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

RNA-seq transcriptome analysis of formalin fixed, paraffin-embedded canine meningioma

PubMed Central

Grenier, Jennifer K.; Foureman, Polly A.; Sloma, Erica A.

2017-01-01

Meningiomas are the most commonly reported primary intracranial tumor in dogs and humans and between the two species there are similarities in histology and biologic behavior. Due to these similarities, dogs have been proposed as models for meningioma pathobiology. However, little is known about specific pathways and individual genes that are involved in the development and progression of canine meningioma. In addition, studies are lacking that utilize RNAseq to characterize gene expression in clinical cases of canine meningioma. The primary objective of this study was to develop a technique for which high quality RNA can be extracted from formalin-fixed, paraffin embedded tissue and then used for transcriptome analysis to determine patterns of gene expression. RNA was extracted from thirteen canine meningiomas–eleven from formalin fixed and two flash-frozen. These represented six grade I and seven grade II meningiomas based on the World Health Organization classification system for human meningioma. RNA was also extracted from fresh frozen leptomeninges from three control dogs for comparison. RNAseq libraries made from formalin fixed tissue were of sufficient quality to successfully identify 125 significantly differentially expressed genes, the majority of which were related to oncogenic processes. Twelve genes (AQP1, BMPER, FBLN2, FRZB, MEDAG, MYC, PAMR1, PDGFRL, PDPN, PECAM1, PERP, ZC2HC1C) were validated using qPCR. Among the differentially expressed genes were oncogenes, tumor suppressors, transcription factors, VEGF-related genes, and members of the WNT pathway. Our work demonstrates that RNA of sufficient quality can be extracted from FFPE canine meningioma samples to provide biologically relevant transcriptome analyses using a next-generation sequencing technique, such as RNA-seq. PMID:29073243

Methylated nucleosides in tRNA and tRNA methyltransferases

PubMed Central

Hori, Hiroyuki

2014-01-01

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

Human blood RNA stabilization in samples collected and transported for a large biobank

PubMed Central

2012-01-01

Background The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999, comprising more than 108.000 pregnancies recruited between 1999 and 2008. In this study we evaluated the feasibility of integrating RNA analyses into existing MoBa protocols. We compared two different blood RNA collection tube systems – the PAXgene™ Blood RNA system and the Tempus™ Blood RNA system - and assessed the effects of suboptimal blood volumes in collection tubes and of transportation of blood samples by standard mail. Endpoints to characterize the samples were RNA quality and yield, and the RNA transcript stability of selected genes. Findings High-quality RNA could be extracted from blood samples stabilized with both PAXgene and Tempus tubes. The RNA yields obtained from the blood samples collected in Tempus tubes were consistently higher than from PAXgene tubes. Higher RNA yields were obtained from cord blood (3 – 4 times) compared to adult blood with both types of tubes. Transportation of samples by standard mail had moderate effects on RNA quality and RNA transcript stability; the overall RNA quality of the transported samples was high. Some unexplained changes in gene expression were noted, which seemed to correlate with suboptimal blood volumes collected in the tubes. Temperature variations during transportation may also be of some importance. Conclusions Our results strongly suggest that special collection tubes are necessary for RNA stabilization and they should be used for establishing new biobanks. We also show that the 50,000 samples collected in the MoBa biobank provide RNA of high quality and in sufficient amounts to allow gene expression analyses for studying the association of disease with altered patterns of gene expression. PMID:22988904

Corn rootworm-active RNA DvSnf7: Repeat dose oral toxicology assessment in support of human and mammalian safety.

PubMed

Petrick, Jay S; Frierdich, Gregory E; Carleton, Stephanie M; Kessenich, Colton R; Silvanovich, Andre; Zhang, Yuanji; Koch, Michael S

2016-11-01

Genetically modified (GM) crops have been developed and commercialized that utilize double stranded RNAs (dsRNA) to suppress a target gene(s), producing virus resistance, nutritional and quality traits. MON 87411 is a GM maize variety that leverages dsRNAs to selectively control corn rootworm through production of a 240 base pair (bp) dsRNA fragment targeting for suppression the western corn rootworm (Diabrotica virgifera virgifera) Snf7 gene (DvSnf7). A bioinformatics assessment found that endogenous corn small RNAs matched ∼450 to 2300 unique RNA transcripts that likely code for proteins in rat, mouse, and human, demonstrating safe dsRNA consumption by mammals. Mice were administered DvSnf7 RNA (968 nucleotides, including the 240 bp DvSnf7 dsRNA) at 1, 10, or 100 mg/kg by oral gavage in a 28-day repeat dose toxicity study. No treatment-related effects were observed in body weights, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. Therefore, the No Observed Adverse Effect Level (NOAEL) for DvSnf7 RNA was 100 mg/kg, the highest dose tested. These results demonstrate that dsRNA for insect control does not produce adverse health effects in mammals at oral doses millions to billions of times higher than anticipated human exposures and therefore poses negligible risk to mammals. Copyright © 2016 Monsanto Company. Published by Elsevier Inc. All rights reserved.

THE MAQC (MICROARRAY QUALITY CONTROL) PROJECT: CALIBRATED RNA SAMPLES, REFERENCE DATASETS, AND QC METRICS AND THRESHOLDS

EPA Science Inventory

FDAs Critical Path Initiative identifies pharmacogenomics and toxicogenomics as key opportunities in advancing medical product development and personalized medicine, and the Guidance for Industry: Pharmacogenomic Data Submissions has been released. Microarrays represent a co...

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

PubMed Central

Murayama, Asako; Sugiyama, Nao; Watashi, Koichi; Masaki, Takahiro; Suzuki, Ryosuke; Aizaki, Hideki; Mizuochi, Toshiaki; Wakita, Takaji

2012-01-01

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits. PMID:22495557

DDX3 DEAD-box RNA helicase plays a central role in mitochondrial protein quality control in Leishmania

PubMed Central

Padmanabhan, Prasad Kottayil; Zghidi-Abouzid, Ouafa; Samant, Mukesh; Dumas, Carole; Aguiar, Bruno Guedes; Estaquier, Jerome; Papadopoulou, Barbara

2016-01-01

DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3's function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control. PMID:27735940

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

PubMed

Boulon, Séverine; Pradet-Balade, Bérengère; Verheggen, Céline; Molle, Dorothée; Boireau, Stéphanie; Georgieva, Marya; Azzag, Karim; Robert, Marie-Cécile; Ahmad, Yasmeen; Neel, Henry; Lamond, Angus I; Bertrand, Edouard

2010-09-24

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases. Copyright © 2010 Elsevier Inc. All rights reserved.

HSP90 and Its R2TP/Prefoldin-like Cochaperone Are Involved in the Cytoplasmic Assembly of RNA Polymerase II

PubMed Central

Boireau, Stéphanie; Georgieva, Marya; Azzag, Karim; Robert, Marie-Cécile; Ahmad, Yasmeen; Neel, Henry; Lamond, Angus I.; Bertrand, Edouard

2015-01-01

SUMMARY RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases. PMID:20864038

Insights into RNA processing pathways and associated-RNA degrading enzymes in Archaea.

PubMed

Clouet-d'Orval, Béatrice; Batista, Manon; Bouvier, Marie; Quentin, Yves; Fichant, Gwennaele; Marchfelder, Anita; Maier, Lisa-Katharina

2018-04-19

RNA processing pathways are at the center of regulation of gene expression. All RNA transcripts undergo multiple maturation steps in addition to covalent chemical modifications to become functional in the cell. This includes destroying unnecessary or defective cellular RNAs. In Archaea, information on mechanisms by which RNA species reach their mature forms and associated RNA-modifying enzymes is still fragmentary. To date, most archaeal actors and pathways have been proposed in light of information gathered from Bacteria and Eukarya. In this context, this review provides a state of the art overview of archaeal endoribonucleases and exoribonucleases that cleave and trim RNA species and also of the key small archaeal proteins that bind RNAs. Furthermore, synthetic up-to-date views of processing and biogenesis pathways of archaeal transfer and ribosomal RNAs as well as of maturation of stable small non-coding RNAs such as CRISPR RNAs, small C/D and H/ACA box guide RNAs, and other emerging classes of small RNAs are described. Finally prospective post-transcriptional mechanisms to control archaeal messenger RNA quality and quantity are discussed.

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

PubMed Central

Kosmaczewski, Sara Guckian; Edwards, Tyson James; Han, Sung Min; Eckwahl, Matthew J; Meyer, Benjamin Isaiah; Peach, Sally; Hesselberth, Jay R; Wolin, Sandra L; Hammarlund, Marc

2014-01-01

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo. Subject Categories Protein Biosynthesis & Quality Control; RNA Biology PMID:25366321

Finding Order in Randomness: Single-Molecule Studies Reveal Stochastic RNA Processing | Center for Cancer Research

Cancer.gov

Producing a functional eukaryotic messenger RNA (mRNA) requires the coordinated activity of several large protein complexes to initiate transcription, elongate nascent transcripts, splice together exons, and cleave and polyadenylate the 3’ end. Kinetic competition between these various processes has been proposed to regulate mRNA maturation, but this model could lead to multiple, randomly determined, or stochastic, pathways or outcomes. Regulatory checkpoints have been suggested as a means of ensuring quality control. However, current methods have been unable to tease apart the contributions of these processes at a single gene or on a time scale that could provide mechanistic insight. To begin to investigate the kinetic relationship between transcription and splicing, Daniel Larson, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues employed a single-molecule RNA imaging approach to monitor production and processing of a human β-globin reporter gene in living cells.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria.

PubMed

Chauhan, Arjun; Sharma, J N; Modgil, Manju; Siddappa, Sundaresha

2018-05-29

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus. Copyright © 2018 Elsevier B.V. All rights reserved.

Intra-Platform Repeatability and Inter-Platform Comparability of MicroRNA Microarray Technology

PubMed Central

Sato, Fumiaki; Tsuchiya, Soken; Terasawa, Kazuya; Tsujimoto, Gozoh

2009-01-01

Over the last decade, DNA microarray technology has provided a great contribution to the life sciences. The MicroArray Quality Control (MAQC) project demonstrated the way to analyze the expression microarray. Recently, microarray technology has been utilized to analyze a comprehensive microRNA expression profiling. Currently, several platforms of microRNA microarray chips are commercially available. Thus, we compared repeatability and comparability of five different microRNA microarray platforms (Agilent, Ambion, Exiqon, Invitrogen and Toray) using 309 microRNAs probes, and the Taqman microRNA system using 142 microRNA probes. This study demonstrated that microRNA microarray has high intra-platform repeatability and comparability to quantitative RT-PCR of microRNA. Among the five platforms, Agilent and Toray array showed relatively better performances than the others. However, the current lineup of commercially available microRNA microarray systems fails to show good inter-platform concordance, probably because of lack of an adequate normalization method and severe divergence in stringency of detection call criteria between different platforms. This study provided the basic information about the performance and the problems specific to the current microRNA microarray systems. PMID:19436744

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

PubMed Central

Liang, Chaoqiong; Hao, Jianjun; Meng, Yan; Luo, Laixin; Li, Jianqiang

2018-01-01

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall. PMID:29543906

MAGI: a Node.js web service for fast microRNA-Seq analysis in a GPU infrastructure.

PubMed

Kim, Jihoon; Levy, Eric; Ferbrache, Alex; Stepanowsky, Petra; Farcas, Claudiu; Wang, Shuang; Brunner, Stefan; Bath, Tyler; Wu, Yuan; Ohno-Machado, Lucila

2014-10-01

MAGI is a web service for fast MicroRNA-Seq data analysis in a graphics processing unit (GPU) infrastructure. Using just a browser, users have access to results as web reports in just a few hours->600% end-to-end performance improvement over state of the art. MAGI's salient features are (i) transfer of large input files in native FASTA with Qualities (FASTQ) format through drag-and-drop operations, (ii) rapid prediction of microRNA target genes leveraging parallel computing with GPU devices, (iii) all-in-one analytics with novel feature extraction, statistical test for differential expression and diagnostic plot generation for quality control and (iv) interactive visualization and exploration of results in web reports that are readily available for publication. MAGI relies on the Node.js JavaScript framework, along with NVIDIA CUDA C, PHP: Hypertext Preprocessor (PHP), Perl and R. It is freely available at http://magi.ucsd.edu. © The Author 2014. Published by Oxford University Press.

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus).

PubMed

Parthipan, Sivashanmugam; Selvaraju, Sellappan; Somashekar, Lakshminarayana; Kolte, Atul P; Arangasamy, Arunachalam; Ravindra, Janivara Parameswaraiah

2015-08-01

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at -80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy+TRIzol and PureLink+TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800-900 ng/30-40 × 10(6)) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50-2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa. Copyright © 2015 Elsevier Inc. All rights reserved.

Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments.

PubMed

Choi, Sungshin; Ray, Hami E; Lai, San-Huei; Alwood, Joshua S; Globus, Ruth K

2016-01-01

Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved. The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C. Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH). Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6-7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months. These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.

Evaluation of isolation methods for bacterial RNA quantitation in Dickeya dadantii

USDA-ARS?s Scientific Manuscript database

Dickeya dadantii is a difficult source for RNA of a sufficient quality for real-time qRT-PCR analysis of gene expression. Three RNA isolation methods were evaluated for their ability to produce high-quality RNA from this bacterium. Bacterial lysis with Trizol using standard protocols consistently ga...

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

PubMed Central

2012-01-01

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings. PMID:16964229

The determinants of alternative RNA splicing in human cells.

PubMed

Ramanouskaya, Tatsiana V; Grinev, Vasily V

2017-12-01

Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

Cbp80 is needed for the expression of piRNA components and piRNAs

PubMed Central

Colombo, Martino; Hernandez, Greco; Beuchle, Dirk; Berger, Fabienne; Peischl, Stephan; Bruggmann, Rémy

2017-01-01

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts. PMID:28746365

Notochord isolation using laser capture microdissection.

PubMed

Santegoeds, R G C; Yakkioui, Y; Jahanshahi, A; Raven, G; Van Overbeeke, J J; Herrler, A; Temel, Y

2017-03-01

Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

PubMed Central

Yan, Xu; Bishop, David J.

2018-01-01

Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content. PMID:29746477

Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing.

PubMed

Riesgo, Ana; Pérez-Porro, Alicia R; Carmona, Susana; Leys, Sally P; Giribet, Gonzalo

2012-03-01

Transcriptome sequencing with next-generation sequencing technologies has the potential for addressing many long-standing questions about the biology of sponges. Transcriptome sequence quality depends on good cDNA libraries, which requires high-quality mRNA. Standard protocols for preserving and isolating mRNA often require optimization for unusual tissue types. Our aim was assessing the efficiency of two preservation modes, (i) flash freezing with liquid nitrogen (LN₂) and (ii) immersion in RNAlater, for the recovery of high-quality mRNA from sponge tissues. We also tested whether the long-term storage of samples at -80 °C affects the quantity and quality of mRNA. We extracted mRNA from nine sponge species and analysed the quantity and quality (A260/230 and A260/280 ratios) of mRNA according to preservation method, storage time, and taxonomy. The quantity and quality of mRNA depended significantly on the preservation method used (LN₂) outperforming RNAlater), the sponge species, and the interaction between them. When the preservation was analysed in combination with either storage time or species, the quantity and A260/230 ratio were both significantly higher for LN₂-preserved samples. Interestingly, individual comparisons for each preservation method over time indicated that both methods performed equally efficiently during the first month, but RNAlater lost efficiency in storage times longer than 2 months compared with flash-frozen samples. In summary, we find that for long-term preservation of samples, flash freezing is the preferred method. If LN₂ is not available, RNAlater can be used, but mRNA extraction during the first month of storage is advised. © 2011 Blackwell Publishing Ltd.

Evaluations of methods for the isolation of high quality RNA from bovine and cervine hide biopsies for use in gene expression studies

USDA-ARS?s Scientific Manuscript database

Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data pr...

Effect of storage time on gene expression data acquired from unfrozen archived newborn blood spots.

PubMed

Ho, Nhan T; Busik, Julia V; Resau, James H; Paneth, Nigel; Khoo, Sok Kean

2016-11-01

Unfrozen archived newborn blood spots (NBS) have been shown to retain sufficient messenger RNA (mRNA) for gene expression profiling. However, the effect of storage time at ambient temperature for NBS samples in relation to the quality of gene expression data is relatively unknown. Here, we evaluated mRNA expression from quantitative real-time PCR (qRT-PCR) and microarray data obtained from NBS samples stored at ambient temperature to determine the effect of storage time on the quality of gene expression. These data were generated in a previous case-control study examining NBS in 53 children with cerebral palsy (CP) and 53 matched controls. NBS sample storage period ranged from 3 to 16years at ambient temperature. We found persistently low RNA integrity numbers (RIN=2.3±0.71) and 28S/18S rRNA ratios (~0) across NBS samples for all storage periods. In both qRT-PCR and microarray data, the expression of three common housekeeping genes-beta cytoskeletal actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA)-decreased with increased storage time. Median values of each microarray probe intensity at log 2 scale also decreased over time. After eight years of storage, probe intensity values were largely reduced to background intensity levels. Of 21,500 genes tested, 89% significantly decreased in signal intensity, with 13,551, 10,730, and 9925 genes detected within 5years, > 5 to <10years, and >10years of storage, respectively. We also examined the expression of two gender-specific genes (X inactivation-specific transcript, XIST and lysine-specific demethylase 5D, KDM5D) and seven gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways hypothesized to be related to CP risk to determine the effect of storage time on the detection of these biologically relevant genes. We found the gender-specific genes and CP-related gene sets detectable in all storage periods, but exhibited differential expression (between male vs. female or CP vs. control) only within the first six years of storage. We concluded that gene expression data quality deteriorates in unfrozen archived NBS over time and that differential gene expression profiling and analysis is recommended for those NBS samples collected and stored within six years at ambient temperature. Copyright © 2016 Elsevier Inc. All rights reserved.

RNA quality in fresh-frozen gastrointestinal tumor specimens-experiences from the tumor and healthy tissue bank TU Dresden.

PubMed

Zeugner, Silke; Mayr, Thomas; Zietz, Christian; Aust, Daniela E; Baretton, Gustavo B

2015-01-01

The term "pre-analytics" summarizes all procedures concerned with specimen collection or processing as well as logistical aspects like transport or storage of tissue specimens. All or these variables as well as tissue-specific characteristics affect sample quality. While certain parameters like warm ischemia or tissue-specific characteristics cannot be changed, other parameters can be assessed and optimized. The aim of this study was to determine RNA quality by assessing the RIN values of specimens from different organs and to assess the influence of vacuum preservation. Samples from the GI tract, in general, appear to have lower RNA quality when compared to samples from other organ sites. This may be due to the digestive enzymes or bacterial colonization. Processing time in pathology does not significantly influence RNA quality. Tissue preservation with a vacuum sealer leads to preserved RNA quality over an extended period of time and offers a feasible alternative to minimize the influence of transport time into pathology.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

EPA Science Inventory

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, ...

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Lai, S.; Klotz, R.; Popova, Y.; Chakravarty, K.; Beegle, J. E.; Wigley, C. L.; Globus, R. K.

2014-01-01

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development; Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science. Mouse livers and spleens were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection operations including storage at -80 C for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RIN(is) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNAlater) and liver (fast-freezing) at various time points post-euthanasia (from 5 min up to 105 min). The RNA recovered was of high quality (spleen, RIN (is) greater than 8; liver, RIN (is) greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at the latest time (105 min). Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) Many additional tissues for gene expression analysis can be obtained by dissection following prolonged storage of the tissue in situ at -80 C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to all future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

PubMed

Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

2015-05-01

Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter?

PubMed

Samadani, Ali Akbar; Nikbakhsh, Novin; Fattahi, Sadegh; Pourbagher, Roghayeh; Aghajanpour Mir, Seyyed Mohsen; Mousavi Kani, Narges; Abedian, Zeinab; Akhavan-Niaki, Haleh

2015-01-01

The reliability of gene expression profiling, based technologies and methods to find transcriptional differences representative of the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was accomplished to optimize the RNA extraction methods and compare the amounts of tissue or quality of tissue. Relatively, the cancerous tissue of human stomach in fresh and frozen conditions and also the mouse fresh tissue were studied. Some factors like the amount of samples, efficacy differences of diverse extraction buffers (TriPure, Trizol) and also the efficacy of b-mercaptoethanol were compared and investigated. The results indicated that the less amount (1-2 mg) compared to other amounts (2-5 mg, 5-15 mg) yielded the best quality and the RNA bands (5S, 18S, 28S) were observed perfectly. Relatively, comparing and measuring some kinds of buffers (Trizol, TriPure) indicated no difference in RNA extraction quality. The last investigated factor was the effect of b- mercaptoethanol which was used along with TriPure to remove the RNAse. Conclusively, no effective impression was observed.

Selenocysteine incorporation: A trump card in the game of mRNA decay

PubMed Central

Shetty, Sumangala P.; Copeland, Paul R.

2015-01-01

The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNASec recognizes a UGA codon. This sets up an intriguing interplay between translation elongation, translation termination and the complex machinery that marks mRNAs that contain premature termination codons for degradation, leading to nonsense mediated mRNA decay (NMD). In this review we discuss the intricate and complex relationship between this key quality control mechanism and the process of Sec incorporation in mammals. PMID:25622574

rnaQUAST: a quality assessment tool for de novo transcriptome assemblies.

PubMed

Bushmanova, Elena; Antipov, Dmitry; Lapidus, Alla; Suvorov, Vladimir; Prjibelski, Andrey D

2016-07-15

Ability to generate large RNA-Seq datasets created a demand for both de novo and reference-based transcriptome assemblers. However, while many transcriptome assemblers are now available, there is still no unified quality assessment tool for RNA-Seq assemblies. We present rnaQUAST-a tool for evaluating RNA-Seq assembly quality and benchmarking transcriptome assemblers using reference genome and gene database. rnaQUAST calculates various metrics that demonstrate completeness and correctness levels of the assembled transcripts, and outputs them in a user-friendly report. rnaQUAST is implemented in Python and is freely available at http://bioinf.spbau.ru/en/rnaquast ap@bioinf.spbau.ru Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Using microRNA profiling in urine samples to develop a non-invasive test for bladder cancer.

PubMed

Mengual, Lourdes; Lozano, Juan José; Ingelmo-Torres, Mercedes; Gazquez, Cristina; Ribal, María José; Alcaraz, Antonio

2013-12-01

Current standard methods used to detect and monitor bladder urothelial cell carcinoma (UCC) are invasive or have low sensitivity. The incorporation into clinical practice of a non-invasive tool for UCC assessment would enormously improve patients' quality of life and outcome. This study aimed to examine the microRNA (miRNA) expression profiles in urines of UCC patients in order to develop a non-invasive accurate and reliable tool to diagnose and provide information on the aggressiveness of the tumor. We performed a global miRNA expression profiling analysis of the urinary cells from 40 UCC patients and controls using TaqMan Human MicroRNA Array followed by validation of 22 selected potentially diagnostic and prognostic miRNAs in a separate cohort of 277 samples using a miRCURY LNA qPCR system. miRNA-based signatures were developed by multivariate logistic regression analysis and internally cross-validated. In the initial cohort of patients, we identified 40 and 30 aberrantly expressed miRNA in UCC compared with control urines and in high compared with low grade tumors, respectively. Quantification of 22 key miRNAs in an independent cohort resulted in the identification of a six miRNA diagnostic signature with a sensitivity of 84.8% and specificity of 86.5% (AUC = 0.92) and a two miRNA prognostic model with a sensitivity of 84.95% and a specificity of 74.14% (AUC = 0.83). Internal cross-validation analysis confirmed the accuracy rates of both models, reinforcing the strength of our findings. Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable tool for the non-invasive assessment of UCC. Copyright © 2013 UICC.

MICROARRAY QUALITY CONTROL PROJECT: A COMPREHENSIVE GENE EXPRESSION TECHNOLOGY SURVEY DEMONSTRATES MEASURABLE CONSISTENCY AND CONCORDANT RESULTS BETWEEN PLATFORMS

EPA Science Inventory

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, h...

Requirements for rapid plasmid ColE1 copy number adjustments: a mathematical model of inhibition modes and RNA turnover rates.

PubMed

Paulsson, J; Nordström, K; Ehrenberg, M

1998-01-01

The random distribution of ColE1 plasmids between the daughter cells at cell division introduces large copy number variations. Statistic variation associated with limited copy number in single cells also causes fluctuations to emerge spontaneously during the cell cycle. Efficient replication control out of steady state is therefore important to tame such stochastic effects of small numbers. In the present model, the dynamic features of copy number control are divided into two parts: first, how sharply the replication frequency per plasmid responds to changes in the concentration of the plasmid-coded inhibitor, RNA I, and second, how tightly RNA I and plasmid concentrations are coupled. Single (hyperbolic)- and multiple (exponential)-step inhibition mechanisms are compared out of steady state and it is shown how the response in replication frequency depends on the mode of inhibition. For both mechanisms, sensitivity of inhibition is "bought" at the expense of a rapid turnover of a replication preprimer, RNA II. Conventional, single-step, inhibition kinetics gives a sloppy replication control even at high RNA II turnover rates, whereas multiple-step inhibition has the potential of working with unlimited precision. When plasmid concentration changes rapidly, RNA I must be degraded rapidly to be "up to date" with the change. Adjustment to steady state is drastically impaired when the turnover rate constants of RNA I decrease below certain thresholds, but is basically unaffected for a corresponding increase. Several features of copy number control that are shown to be crucial for the understanding of ColE1-type plasmids still remain to be experimentally characterized. It is shown how steady-state properties reflect dynamics at the heart of regulation and therefore can be used to discriminate between fundamentally different copy number control mechanisms. The experimental tests of the predictions made require carefully planned assays, and some suggestions for suitable experiments arise naturally from the present work. It is also discussed how the presence of the Rom protein may affect dynamic qualities of copy number control. Copyright 1998 Academic Press.

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

PubMed

Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

2016-02-04

Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

Improving molecular tools for global surveillance of measles virus.

PubMed

Bankamp, Bettina; Byrd-Leotis, Lauren A; Lopareva, Elena N; Woo, Gibson K S; Liu, Chunyu; Jee, Youngmee; Ahmed, Hinda; Lim, Wilina W; Ramamurty, Nalini; Mulders, Mick N; Featherstone, David; Bellini, William J; Rota, Paul A

2013-09-01

The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. Published by Elsevier B.V.

The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder.

PubMed

Liu, Lu; Mansfield, Kylie J; Kristiana, Ika; Vaux, Kenneth J; Millard, Richard J; Burcher, Elizabeth

2007-01-01

Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.

Improving molecular tools for global surveillance of measles virus⋆

PubMed Central

Bankamp, Bettina; Byrd-Leotis, Lauren A.; Lopareva, Elena N.; Woo, Gibson K.S.; Liu, Chunyu; Jee, Youngmee; Ahmed, Hinda; Lim, Wilina W.; Ramamurty, Nalini; Mulders, Mick N.; Featherstone, David; Bellini, William J.; Rota, Paul A.

2017-01-01

Background The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. Objectives The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. Study design A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. Results A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. Conclusions These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures. PMID:23806666

Hippo pathway deficiency reverses systolic heart failure after infarction.

PubMed

Leach, John P; Heallen, Todd; Zhang, Min; Rahmani, Mahdis; Morikawa, Yuka; Hill, Matthew C; Segura, Ana; Willerson, James T; Martin, James F

2017-10-12

Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration, is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2. Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

PubMed Central

Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

2016-01-01

Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels. PMID:27575051

Muscle cell atrophy induced by HSP gene silencing was counteracted by HSP overexpression

NASA Astrophysics Data System (ADS)

Choi, Inho; Lee, Joo-Hee; Nikawa, Takeshi; Gwag, Taesik; Park, Kyoungsook; Park, Junsoo

Heat shock proteins (HSP), as molecular chaperones, are known to assist protein quality control under various stresses. Although overexpression of HSP70 was found to contribute to muscle size retention under an unloading condition, it remains largely unclarified whether muscle atrophy is induced by active suppression of HSP expression. In this study, we pre-treated Hsp70 siRNA to rat L6 cells for the HSP gene silencing, and determined myotube diameter, HSP72 expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (CEL), the HSP70 inducer. Relative to a negative control (NC), muscle cell diameter was reduced 0.89-fold in the siRNA-treated group, increased 1.2-fold in the CEL-treated group and retained at the size of NC in the siRNA+CEL group. HSP72 expression was decreased 0.35-fold by siRNA whereas the level was increased 6- to 8-fold in the CEL and siRNA+CEL groups. Expression of FoxO3 and atrogin-1 was increased 1.8- to 4.8-fold by siRNA, which was abolished by CEL treatment. Finally, phosphorylation of Akt1, S6K and ERK1/2 was not affected by siRNA, but was elevated 2- to 6-fold in the CEL and siRNA+CEL groups. Taken together, HSP downregulation by Hsp gene silencing led to muscle cell atrophy principally via increases in catabolic activities and that such anti-atrophic effect was counteracted by HSP overexpression.

Structures of the tRNA export factor in the nuclear and cytosolic states.

PubMed

Cook, Atlanta G; Fukuhara, Noemi; Jinek, Martin; Conti, Elena

2009-09-03

Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-beta family (Xpot, also known as Los1 in Saccharomyces cerevisiae). Here we report the 3.2 A resolution structure of Schizosaccharomyces pombe Xpot in complex with tRNA and RanGTP, and the 3.1 A structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5' and 3' ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control.

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

PubMed Central

Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

2015-01-01

In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

MicroRNA Changes in Cerebrospinal Fluid After Subarachnoid Hemorrhage.

PubMed

Bache, Søren; Rasmussen, Rune; Rossing, Maria; Laigaard, Finn Pedersen; Nielsen, Finn Cilius; Møller, Kirsten

2017-09-01

Delayed cerebral ischemia (DCI) accounts for a major part of the morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). MicroRNAs (miRNAs) are pathophysiologically involved in acute cerebral ischemia. This study compared miRNA profiles in cerebrospinal fluid from neurologically healthy patients, as well as SAH patients with and without subsequent development of DCI. In a prospective case-control study of SAH patients treated with external ventricular drainage and neurologically healthy patients, miRNA profiles in cerebrospinal fluid were screened and validated using 2 different high-throughput real-time quantification polymerase chain reaction techniques. The occurrence of DCI was documented in patient charts and subsequently reviewed independently by 2 physicians. MiRNA profiles from 27 SAH patients and 10 neurologically healthy patients passed quality control. In the validation, 66 miRNAs showed a relative increase in cerebrospinal fluid from SAH patients compared with neurologically healthy patients ( P <0.001); 2 (miR-21 and miR-221) showed a relative increase in SAH patients with DCI compared with those without ( P <0.05) in both the screening and validation. SAH is associated with marked changes in the cerebrospinal fluid miRNA profile. These changes could be associated to the development of DCI. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01791257. © 2017 The Authors.

Field-based evaluation of a male-specific (F+) RNA coliphage ...

EPA Pesticide Factsheets

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Thus, sensitive, reliable and easy to use methods for the detection of microorganisms are needed to evaluate water quality. In this study, we performed a field evaluation of an anion-exchange resin based platform to concentrate F-RNA coliphages (fecal/enteric virus indicators) from diverse fecally impacted environmental waters. In this platform, F-RNA coliphages are adsorbed to anion-exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase PCR detection. Matrix-dependent inhibition was evaluated using known quantities of spiked F-RNA coliphage genogroups GI, GII, GII and GIV. Detection was successful in 97%, 72%, 85% and 98% of the samples for spiked F-RNA coliphage GI, GII, GIII and GIV, respectively, and was differentially affected by inhibitory properties specific to each water sample. No association between inhibition and the water samples’ physicochemical properties was apparent. Parallel evaluations of the spiked samples with internal amplification control (IAC) reactions (a widely used control to assess inhibition) demonstrated that IAC reaction inhibition was not agreement with that observed for spiked samples, suggesting that testing of spiked samples allows for better assessments of matrix-dependent inhibition. Additionally, the anion-

Selection of Reference Gene Expression in a Schizophrenia Brain Cohort

PubMed Central

Weickert, Cynthia Shannon; Sheedy, Donna; Rothmond, Debora A.; Dedova, Irina; Fung, Samantha; Garrick, Therese; Wong, Jenny; Harding, Antony J.; Sivagnanansundaram, Sinthuja; Hunt, Clare; Duncan, Carlotta; Sundqvist, Nina; Tsai, Shan-Yuan; Anand, Jasna; Draganic, Daren; Harper, Clive

2010-01-01

Objective To conduct postmortem human brain research into the neuropathological basis of schizophrenia, it is critical to establish cohorts that are well-characterised and well-matched. Our objective was to determine if specimen characteristics, including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs. Methods We selected a matched cohort of 74 cases (37 schizophrenia / schizoaffective disorder cases and 37 controls cases). Middle frontal gyrus tissue was pulverised, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using RT-PCR, we measured nine housekeeper genes and calculated a geomean in each diagnostic group. Results We found that the RINs were very good (mean 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH, and two clinical variables, agonal state and duration of illness did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. Our results show that people with schizophrenia had significantly less PPIA, and SDHA and tended to have less GUSB and B2M mRNA suggesting that these control genes may not be good candidates for normalisation. Conclusions In our cohort, less than 10% variability in RIN values was detected and the diagnostic groups were well matched overall. Our cohort was adequately powered (0.80–0.90) to detect mRNA differences (25%) due to disease. Our study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected. PMID:20073568

[Evaluation of quality of HIV diagnostic procedures in Poland].

PubMed

Parczewski, Miłosz; Madaliński, Kazimierz; Leszczyszyn-Pynka, Magdalena; Boroń-Kaczmarska, Anna

2010-01-01

The aim of this work was quality assessment of HIV diagnostic procedures in Poland, including human and technical resources as well as laboratory practice. Sixty questionnaires were distributed among diagnostic centers to obtain qualitative data. Basing on the survey data serological control using coded panels of HIV-1/2 samples was performed. Thirty-one filled questionnaires were received (50.8%). Surveyed laboratories perform from 350 to 5500 serological screening tests per year. In most of laboratories fourth generation assays are available, while Blood Donation Centers screen the blood both with serological assays and by HIV-RNA detection. Sanitary and Epidemiological Stations and academic laboratories hold the ISO/IEC 17025 or IS0 9001:2001 accreditation, five of the surveyed centers participate in Labquality assurance and two in Quality Control in Molecular Diagnostics programs. Data of control serological testing were received from 21 centers. In the quality control assessment 194 analyses were performed with 91 true negative, 2 false negative, 96 true positive and 5 false positive results. False negative rate of % and false positive rate of 5.2% was noted for this study. Currently, virtually no guidelines related to the HIV-diagnostics quality assurance and control in Poland are in delineated. Development of the national unified quality control system, basing on the central institution is highly desirable. National certification within the frames of the quality control and assurance program should be mandatory for all the diagnostic labs, and aim at improvement of reliability of the result distributed among clinicians and patients.

PeTMbase: A Database of Plant Endogenous Target Mimics (eTMs).

PubMed

Karakülah, Gökhan; Yücebilgili Kurtoğlu, Kuaybe; Unver, Turgay

2016-01-01

MicroRNAs (miRNA) are small endogenous RNA molecules, which regulate target gene expression at post-transcriptional level. Besides, miRNA activity can be controlled by a newly discovered regulatory mechanism called endogenous target mimicry (eTM). In target mimicry, eTMs bind to the corresponding miRNAs to block the binding of specific transcript leading to increase mRNA expression. Thus, miRNA-eTM-target-mRNA regulation modules involving a wide range of biological processes; an increasing need for a comprehensive eTM database arose. Except miRSponge with limited number of Arabidopsis eTM data no available database and/or repository was developed and released for plant eTMs yet. Here, we present an online plant eTM database, called PeTMbase (http://petmbase.org), with a highly efficient search tool. To establish the repository a number of identified eTMs was obtained utilizing from high-throughput RNA-sequencing data of 11 plant species. Each transcriptome libraries is first mapped to corresponding plant genome, then long non-coding RNA (lncRNA) transcripts are characterized. Furthermore, additional lncRNAs retrieved from GREENC and PNRD were incorporated into the lncRNA catalog. Then, utilizing the lncRNA and miRNA sources a total of 2,728 eTMs were successfully predicted. Our regularly updated database, PeTMbase, provides high quality information regarding miRNA:eTM modules and will aid functional genomics studies particularly, on miRNA regulatory networks.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment.

PubMed

Gierliński, Marek; Cole, Christian; Schofield, Pietà; Schurch, Nicholas J; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J

2015-11-15

High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of 'bad' replicates, which can drastically affect the gene read-count distribution. RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. g.j.barton@dundee.ac.uk. © The Author 2015. Published by Oxford University Press.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment

PubMed Central

Cole, Christian; Schofield, Pietà; Schurch, Nicholas J.; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J.

2015-01-01

Motivation: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. Results: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of ‘bad’ replicates, which can drastically affect the gene read-count distribution. Availability and implementation: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. Contact: g.j.barton@dundee.ac.uk PMID:26206307

Circulating microRNA Biomarkers as Liquid Biopsy for Cancer Patients: Pros and Cons of Current Assays

PubMed Central

Ono, Shigeshi; Lam, Stella; Nagahara, Makoto; Hoon, Dave S. B.

2015-01-01

An increasing number of studies have focused on circulating microRNAs (cmiRNA) in cancer patients’ blood for their potential as minimally-invasive biomarkers. Studies have reported the utility of assessing specific miRNAs in blood as diagnostic/prognostic biomarkers; however, the methodologies are not validated or standardized across laboratories. Unfortunately, there is often minimum limited overlap in techniques between results reported even in similar type studies on the same cancer. This hampers interpretation and reliability of cmiRNA as potential cancer biomarkers. Blood collection and processing, cmiRNA extractions, quality and quantity control of assays, defined patient population assessment, reproducibility, and reference standards all affect the cmiRNA assay results. To date, there is no reported definitive method to assess cmiRNAs. Therefore, appropriate and reliable methodologies are highly necessary in order for cmiRNAs to be used in regulated clinical diagnostic laboratories. In this review, we summarize the developments made over the past decade towards cmiRNA detection and discuss the pros and cons of the assays. PMID:26512704

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

PubMed

Stein, Erica V; Duewer, David L; Farkas, Natalia; Romsos, Erica L; Wang, Lili; Cole, Kenneth D

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR

PubMed Central

Duewer, David L.; Farkas, Natalia; Romsos, Erica L.; Wang, Lili; Cole, Kenneth D.

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values. PMID:29145448

Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing

PubMed Central

Tourlousse, Dieter M.; Yoshiike, Satowa; Ohashi, Akiko; Matsukura, Satoko; Noda, Naohiro

2017-01-01

Abstract High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification. PMID:27980100

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

The effect of active immunization against vasoactive intestinal peptide (VIP) and inhibin on reproductive performance of aging White Leghorn roosters.

PubMed

Avital-Cohen, N; Heiblum, R; Argov, N; Rosenstrauch, A; Chaiseha, Y; Mobarkey, N; Rozenboim, I

2012-01-01

Decreasing fertility in aging domestic roosters is a well-known phenomenon. Aging is manifested by a decrease in plasma testosterone level, testis function, and spermatogenesis, resulting in a low level of fertility. The roles of vasoactive intestinal peptide (VIP) and testicular inhibin in this aging process are not clear. The effects of active immunization against VIP, inhibin, or the combination of both hormones on the reproduction of aging White Leghorn (WL) roosters were assayed. In experiment 1a, 60 White Leghorn roosters (67 wk of age) were divided into 4 groups (n = 15/group). The first group was actively immunized against VIP, the second against inhibin, the third against VIP and inhibin, and the fourth served as a control. Active immunization against VIP decreased semen quality parameters, plasma steroid levels, and gene expression of gonadotropin-releasing hormone-I (GnRH-I), follicle-stimulating hormone (FSH), luteinizing hormone (LH), LH receptor, VIP, and prolactin (Prl). Immunization against inhibin increased some of the semen quality parameters and FSH mRNA gene expression but decreased inhibin gene expression. In experiment 1b, at 94 wk of age, we took the actively immunized against VIP group and the control group and divided them into 2 subgroups (n = 7 or 8): the first group was injected with 1 mg of ovine Prl (oPrl) daily for 7 d, and the second group served as a control. Administration of oPrl to previously VIP-immunized birds significantly elevated semen quality parameters. We suggest that VIP, Prl, and inhibin have an important effect on the reproductive axis in aging roosters. Active immunization against VIP-depressed reproductive activity and Prl administration restored their reproduction, indicating that both VIP and Prl are essential for reproduction in aging roosters. Immunization against inhibin improved FSH mRNA gene expression, suggesting a negative role of inhibin on FSH secretion in aging roosters. Not all semen quality parameters increased significantly after immunization against inhibin, even though FSH mRNA gene expression increased, suggesting interference in testicular function in aging roosters.

A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples

PubMed Central

Esteve-Codina, Anna; Arpi, Oriol; Martinez-García, Maria; Pineda, Estela; Mallo, Mar; Gut, Marta; Carrato, Cristina; Rovira, Anna; Lopez, Raquel; Tortosa, Avelina; Dabad, Marc; Del Barco, Sonia; Heath, Simon; Bagué, Silvia; Ribalta, Teresa; Alameda, Francesc; de la Iglesia, Nuria

2017-01-01

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved. PMID:28122052

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

PubMed Central

Beld, Marcel; Minnaar, René; Weel, Jan; Sol, Cees; Damen, Marjolein; van der Avoort, Harry; Wertheim-van Dillen, Pauline; Breda, Alex van; Boom, René

2004-01-01

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens. PMID:15243060

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

PubMed

Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene

2015-07-04

The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples. Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7% for SuperfolderGFP and 14.7-17.8% for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category "Carbohydrate transport and metabolism". Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied.

A Guide for Designing and Analyzing RNA-Seq Data.

PubMed

Chatterjee, Aniruddha; Ahn, Antonio; Rodger, Euan J; Stockwell, Peter A; Eccles, Michael R

2018-01-01

The identity of a cell or an organism is at least in part defined by its gene expression and therefore analyzing gene expression remains one of the most frequently performed experimental techniques in molecular biology. The development of the RNA-Sequencing (RNA-Seq) method allows an unprecedented opportunity to analyze expression of protein-coding, noncoding RNA and also de novo transcript assembly of a new species or organism. However, the planning and design of RNA-Seq experiments has important implications for addressing the desired biological question and maximizing the value of the data obtained. In addition, RNA-Seq generates a huge volume of data and accurate analysis of this data involves several different steps and choices of tools. This can be challenging and overwhelming, especially for bench scientists. In this chapter, we describe an entire workflow for performing RNA-Seq experiments. We describe critical aspects of wet lab experiments such as RNA isolation, library preparation and the initial design of an experiment. Further, we provide a step-by-step description of the bioinformatics workflow for different steps involved in RNA-Seq data analysis. This includes power calculations, setting up a computational environment, acquisition and processing of publicly available data if desired, quality control measures, preprocessing steps for the raw data, differential expression analysis, and data visualization. We particularly mention important considerations for each step to provide a guide for designing and analyzing RNA-Seq data.

Accurate identification of RNA editing sites from primitive sequence with deep neural networks.

PubMed

Ouyang, Zhangyi; Liu, Feng; Zhao, Chenghui; Ren, Chao; An, Gaole; Mei, Chuan; Bo, Xiaochen; Shu, Wenjie

2018-04-16

RNA editing is a post-transcriptional RNA sequence alteration. Current methods have identified editing sites and facilitated research but require sufficient genomic annotations and prior-knowledge-based filtering steps, resulting in a cumbersome, time-consuming identification process. Moreover, these methods have limited generalizability and applicability in species with insufficient genomic annotations or in conditions of limited prior knowledge. We developed DeepRed, a deep learning-based method that identifies RNA editing from primitive RNA sequences without prior-knowledge-based filtering steps or genomic annotations. DeepRed achieved 98.1% and 97.9% area under the curve (AUC) in training and test sets, respectively. We further validated DeepRed using experimentally verified U87 cell RNA-seq data, achieving 97.9% positive predictive value (PPV). We demonstrated that DeepRed offers better prediction accuracy and computational efficiency than current methods with large-scale, mass RNA-seq data. We used DeepRed to assess the impact of multiple factors on editing identification with RNA-seq data from the Association of Biomolecular Resource Facilities and Sequencing Quality Control projects. We explored developmental RNA editing pattern changes during human early embryogenesis and evolutionary patterns in Drosophila species and the primate lineage using DeepRed. Our work illustrates DeepRed's state-of-the-art performance; it may decipher the hidden principles behind RNA editing, making editing detection convenient and effective.

miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

PubMed

Fischer, Simon; Paul, Albert Jesuran; Wagner, Andreas; Mathias, Sven; Geiss, Melanie; Schandock, Franziska; Domnowski, Martin; Zimmermann, Jörg; Handrick, René; Hesse, Friedemann; Otte, Kerstin

2015-10-01

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells. © 2015 Wiley Periodicals, Inc.

Stability of hepatitis C virus RNA and anti-HCV antibody in air-dried and freeze-dried human plasma samples.

PubMed

Poe, Amanda; Duong, Ngocvien Thi; Bedi, Kanwar; Kodani, Maja

2018-03-01

Diagnosis of hepatitis C virus (HCV) infection is based on testing for antibodies to HCV (anti-HCV), hepatitis C core antigen (HCV cAg) and HCV RNA. To ensure quality control (QC) and quality assurance (QA), proficiency panels are provided by reference laboratories and various international organizations, requiring costly dry ice shipments to maintain specimen integrity. Alternative methods of specimen preservation and transport can save on shipping and handling and help in improving diagnostics by facilitating QA/QC of various laboratories especially in resource limited countries. Plasma samples positive for anti-HCV and HCV RNA were either dried using dried tube specimens (DTS) method or lyophilization for varying durations of time and temperature. Preservation of samples using DTS method resulted in loss of anti-HCV reactivity for low-positive samples and did not generate enough volume for HCV RNA testing. Lyophilized samples tested positive for anti-HCV even after storage at 4 °C and 25 °C for 12 weeks. Further, HCV RNA was detectable in 5 of 5 (100%) samples over the course of 12 week storage at 4, 25, 37 and 45 °C. In conclusion, lyophilization of specimens maintains integrity of plasma samples for testing for markers of HCV infection and can be a potent mode of sharing proficiency samples without incurring huge shipping costs and avoids challenges with dry ice shipments between donor and recipient laboratories. Copyright © 2017. Published by Elsevier B.V.

Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture.

PubMed

Nandan, Devki; Thomas, Sneha A; Nguyen, Anne; Moon, Kyung-Mee; Foster, Leonard J; Reiner, Neil E

2017-01-01

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.

Stabilising the Integrity of Snake Venom mRNA Stored under Tropical Field Conditions Expands Research Horizons

PubMed Central

Logan, Rhiannon A. E.; Leung, Kam-Yin D.; Newberry, Fiona J.; Rowley, Paul D.; Dunbar, John P.; Wagstaff, Simon C.; Casewell, Nicholas R.; Harrison, Robert A.

2016-01-01

Background Snake venoms contain many proteinaceous toxins that can cause severe pathology and mortality in snakebite victims. Interestingly, mRNA encoding such toxins can be recovered directly from venom, although yields are low and quality is unknown. It also remains unclear whether such RNA contains information about toxin isoforms and whether it is representative of mRNA recovered from conventional sources, such as the venom gland. Answering these questions will address the feasibility of using venom-derived RNA for future research relevant to biomedical and antivenom applications. Methodology/Principal Findings Venom was extracted from several species of snake, including both members of the Viperidae and Elapidae, and either lyophilized or immediately added to TRIzol reagent. TRIzol-treated venom was incubated at a range of temperatures (4–37°C) for a range of durations (0–48 hours), followed by subsequent RNA isolation and assessments of RNA quantity and quality. Subsequently, full-length toxin transcripts were targeted for PCR amplification and Sanger sequencing. TRIzol-treated venom yielded total RNA of greater quantity and quality than lyophilized venom, and with quality comparable to venom gland-derived RNA. Full-length sequences from multiple Viperidae and Elapidae toxin families were successfully PCR amplified from TRIzol-treated venom RNA. We demonstrated that venom can be stored in TRIzol for 48 hours at 4–19°C, and 8 hours at 37°C, at minimal cost to RNA quality, and found that venom RNA encoded multiple toxin isoforms that seemed homologous (98–99% identity) to those found in the venom gland. Conclusions/Significance The non-invasive experimental modifications we propose will facilitate the future investigation of venom composition by using venom as an alternative source to venom gland tissue for RNA-based studies, thus obviating the undesirable need to sacrifice snakes for such research purposes. In addition, they expand research horizons to rare, endangered or protected snake species and provide more flexibility to performing fieldwork on venomous snakes in tropical conditions. PMID:27280729

Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.

2003-01-01

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

Establishment of the Ph. Eur. Hepatitis A virus RNA for NAT testing BRP batch 1.

PubMed

Chudy, M; Nübling, C M; Blümel, J; Daas, A; Costanzo, A

2017-01-01

Detection of viral contamination in plasma donations is critical to prevent transmission of infectious diseases. The European Pharmacopoeia (Ph. Eur.) monograph 1646 'Human plasma (pooled and treated for virus inactivation)', requires that plasma pools used for the manufacture of this product be tested, among others, for the presence of hepatitis A virus RNA by nucleic acid testing (NAT) using a positive control containing 100 International Units (IU) of hepatitis A virus (HAV) RNA per mL. To this end, the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) organised an international collaborative study under the aegis of the Biological Standardisation Programme, for the establishment of the 1 st Biological Reference Preparation (BRP) for HAV RNA for NAT testing. A freeze-dried candidate material was thus prepared and calibrated against the WHO 2 nd International Standard for HAV for NAT (00/562) in a study in which thirteen European and North American laboratories including Official Medicines Control Laboratories (OMCLs), manufacturers of plasma-derived products, producers of in vitro diagnostic kits and a blood transfusion centre participated. Based on the outcome of the study, an HAV RNA content of 40 000 IU/vial (corresponding approximately to 4.6 log 10 IU/vial) was assigned to the BRP, which was adopted by the Ph. Eur. Commission in March 2016 as Ph. Eur. hepatitis A virus RNA for NAT testing BRP batch 1.

Neuropathological and transcriptomic characteristics of the aged brain

PubMed Central

Miller, Jeremy A; Guillozet-Bongaarts, Angela; Gibbons, Laura E; Postupna, Nadia; Renz, Anne; Beller, Allison E; Sunkin, Susan M; Ng, Lydia; Rose, Shannon E; Smith, Kimberly A; Szafer, Aaron; Barber, Chris; Bertagnolli, Darren; Bickley, Kristopher; Brouner, Krissy; Caldejon, Shiella; Chapin, Mike; Chua, Mindy L; Coleman, Natalie M; Cudaback, Eiron; Cuhaciyan, Christine; Dalley, Rachel A; Dee, Nick; Desta, Tsega; Dolbeare, Tim A; Dotson, Nadezhda I; Fisher, Michael; Gaudreault, Nathalie; Gee, Garrett; Gilbert, Terri L; Goldy, Jeff; Griffin, Fiona; Habel, Caroline; Haradon, Zeb; Hejazinia, Nika; Hellstern, Leanne L; Horvath, Steve; Howard, Kim; Howard, Robert; Johal, Justin; Jorstad, Nikolas L; Josephsen, Samuel R; Kuan, Chihchau L; Lai, Florence; Lee, Eric; Lee, Felix; Lemon, Tracy; Li, Xianwu; Marshall, Desiree A; Melchor, Jose; Mukherjee, Shubhabrata; Nyhus, Julie; Pendergraft, Julie; Potekhina, Lydia; Rha, Elizabeth Y; Rice, Samantha; Rosen, David; Sapru, Abharika; Schantz, Aimee; Shen, Elaine; Sherfield, Emily; Shi, Shu; Sodt, Andy J; Thatra, Nivretta; Tieu, Michael; Wilson, Angela M; Montine, Thomas J; Larson, Eric B; Bernard, Amy; Crane, Paul K; Ellenbogen, Richard G

2017-01-01

As more people live longer, age-related neurodegenerative diseases are an increasingly important societal health issue. Treatments targeting specific pathologies such as amyloid beta in Alzheimer’s disease (AD) have not led to effective treatments, and there is increasing evidence of a disconnect between traditional pathology and cognitive abilities with advancing age, indicative of individual variation in resilience to pathology. Here, we generated a comprehensive neuropathological, molecular, and transcriptomic characterization of hippocampus and two regions cortex in 107 aged donors (median = 90) from the Adult Changes in Thought (ACT) study as a freely-available resource (http://aging.brain-map.org/). We confirm established associations between AD pathology and dementia, albeit with increased, presumably aging-related variability, and identify sets of co-expressed genes correlated with pathological tau and inflammation markers. Finally, we demonstrate a relationship between dementia and RNA quality, and find common gene signatures, highlighting the importance of properly controlling for RNA quality when studying dementia. PMID:29120328

Primer and platform effects on 16S rRNA tag sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tremblay, Julien; Singh, Kanwar; Fern, Alison

Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

Primer and platform effects on 16S rRNA tag sequencing

DOE PAGES

Tremblay, Julien; Singh, Kanwar; Fern, Alison; ...

2015-08-04

Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

Antisense long non-coding RNAs in rainbow trout: Discovery and potential role in muscle growth and quality traits

USDA-ARS?s Scientific Manuscript database

Endogenous mRNA-antisense transcripts are involved in regulation of a wide range of biological processes including muscle development and quality traits of farm animals. Standard RNA-Seq can be used to identify sense-antisense transcripts. However, strand-specific RNA-Seq is required to resolve ambi...

Linking nuclear mRNP assembly and cytoplasmic destiny.

PubMed

Kuersten, Scott; Goodwin, Elizabeth B

2005-06-01

From the very beginning, mRNAs have a complex existence. They are transcribed, capped, spliced, modified at the 3'end, exported from the nucleus, translated, and eventually degraded. These many events not only affect the overall survival and properties of an mRNA, but are also carefully co-ordinated and integrated with quality control mechanisms that function to ensure that only 'proper' mRNAs are translated at the correct developmental time and place. This does not mean that all mRNAs follow a single or uniform path from synthesis to death. Instead, there are diverse means by which the activities of specific mRNAs are regulated, and these controls often depend upon multiple events in the mRNA's life. mRNAs are not found naked in the cell, instead they are part of complex RNPs (ribonucleoproteins) that consist of many factors. These RNPs are highly dynamic structures that change during the lifetime of a given RNA; linking events such as synthesis and processing to the final fate of the mRNA. Here, we will discuss what is known of the assembly of RNPs in general, with specific reference to the myriad of connections between different nuclear events and the cytoplasmic activity of an mRNA. Due to space limitations this review is not comprehensive, instead we focus on specific examples to illustrate these emerging themes in gene expression.

MicroRNA Changes in Cerebrospinal Fluid After Subarachnoid Hemorrhage

PubMed Central

Rasmussen, Rune; Rossing, Maria; Laigaard, Finn Pedersen; Nielsen, Finn Cilius; Møller, Kirsten

2017-01-01

Background and Purpose— Delayed cerebral ischemia (DCI) accounts for a major part of the morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). MicroRNAs (miRNAs) are pathophysiologically involved in acute cerebral ischemia. This study compared miRNA profiles in cerebrospinal fluid from neurologically healthy patients, as well as SAH patients with and without subsequent development of DCI. Methods— In a prospective case–control study of SAH patients treated with external ventricular drainage and neurologically healthy patients, miRNA profiles in cerebrospinal fluid were screened and validated using 2 different high-throughput real-time quantification polymerase chain reaction techniques. The occurrence of DCI was documented in patient charts and subsequently reviewed independently by 2 physicians. Results— MiRNA profiles from 27 SAH patients and 10 neurologically healthy patients passed quality control. In the validation, 66 miRNAs showed a relative increase in cerebrospinal fluid from SAH patients compared with neurologically healthy patients (P<0.001); 2 (miR-21 and miR-221) showed a relative increase in SAH patients with DCI compared with those without (P<0.05) in both the screening and validation. Conclusions— SAH is associated with marked changes in the cerebrospinal fluid miRNA profile. These changes could be associated to the development of DCI. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01791257. PMID:28768799

Mapping by sequencing in cotton (Gossypium hirsutum) line MD52ne identified candidate genes for fiber strength and its related quality attributes.

PubMed

Islam, Md S; Zeng, Linghe; Thyssen, Gregory N; Delhom, Christopher D; Kim, Hee Jin; Li, Ping; Fang, David D

2016-06-01

Three QTL regions controlling three fiber quality traits were validated and further fine-mapped with 27 new single nucleotide polymorphism (SNP) markers. Transcriptome analysis suggests that receptor-like kinases found within the validated QTLs are potential candidate genes responsible for superior fiber strength in cotton line MD52ne. Fiber strength, length, maturity and fineness determine the market value of cotton fibers and the quality of spun yarn. Cotton fiber strength has been recognized as a critical quality attribute in the modern textile industry. Fine mapping along with quantitative trait loci (QTL) validation and candidate gene prediction can uncover the genetic and molecular basis of fiber quality traits. Four previously-identified QTLs (qFBS-c3, qSFI-c14, qUHML-c14 and qUHML-c24) related to fiber bundle strength, short fiber index and fiber length, respectively, were validated using an F3 population that originated from a cross of MD90ne × MD52ne. A group of 27 new SNP markers generated from mapping-by-sequencing (MBS) were placed in QTL regions to improve and validate earlier maps. Our refined QTL regions spanned 4.4, 1.8 and 3.7 Mb of physical distance in the Gossypium raimondii reference genome. We performed RNA sequencing (RNA-seq) of 15 and 20 days post-anthesis fiber cells from MD52ne and MD90ne and aligned reads to the G. raimondii genome. The QTL regions contained 21 significantly differentially expressed genes (DEGs) between the two near-isogenic parental lines. SNPs that result in non-synonymous substitutions to amino acid sequences of annotated genes were identified within these DEGs, and mapped. Taken together, transcriptome and amino acid mutation analysis indicate that receptor-like kinase pathway genes are likely candidates for superior fiber strength and length in MD52ne. MBS along with RNA-seq demonstrated a powerful strategy to elucidate candidate genes for the QTLs that control complex traits in a complex genome like tetraploid upland cotton.

VIPER: Visualization Pipeline for RNA-seq, a Snakemake workflow for efficient and complete RNA-seq analysis.

PubMed

Cornwell, MacIntosh; Vangala, Mahesh; Taing, Len; Herbert, Zachary; Köster, Johannes; Li, Bo; Sun, Hanfei; Li, Taiwen; Zhang, Jian; Qiu, Xintao; Pun, Matthew; Jeselsohn, Rinath; Brown, Myles; Liu, X Shirley; Long, Henry W

2018-04-12

RNA sequencing has become a ubiquitous technology used throughout life sciences as an effective method of measuring RNA abundance quantitatively in tissues and cells. The increase in use of RNA-seq technology has led to the continuous development of new tools for every step of analysis from alignment to downstream pathway analysis. However, effectively using these analysis tools in a scalable and reproducible way can be challenging, especially for non-experts. Using the workflow management system Snakemake we have developed a user friendly, fast, efficient, and comprehensive pipeline for RNA-seq analysis. VIPER (Visualization Pipeline for RNA-seq analysis) is an analysis workflow that combines some of the most popular tools to take RNA-seq analysis from raw sequencing data, through alignment and quality control, into downstream differential expression and pathway analysis. VIPER has been created in a modular fashion to allow for the rapid incorporation of new tools to expand the capabilities. This capacity has already been exploited to include very recently developed tools that explore immune infiltrate and T-cell CDR (Complementarity-Determining Regions) reconstruction abilities. The pipeline has been conveniently packaged such that minimal computational skills are required to download and install the dozens of software packages that VIPER uses. VIPER is a comprehensive solution that performs most standard RNA-seq analyses quickly and effectively with a built-in capacity for customization and expansion.

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA ▿

PubMed Central

Liang, Zhanbei; Keeley, Ann

2011-01-01

Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102 oocysts g−1 of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104 C. parvum oocysts g−1 soil for sandy, loamy, and clay samples, respectively. PMID:21803904

Effect of RNA Integrity Determined With the Agilent 2100 Bioanalyzer on Bacterial RNA Quantification with RT-PCR

USDA-ARS?s Scientific Manuscript database

RNA integrity is critical for successful RNA quantification. The level of integrity required differs among sources and extraction procedures and has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from D. dadantii. The i...

Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

PubMed Central

Olsvik, Pål A; Lie, Kai K; Hevrøy, Ernst M

2007-01-01

Background The aim of the current examination was to evaluate if sedation and anesthetic treatment techniques affect the quality of RNA extracted from liver, gill, head kidney and brain tissues in Atlantic salmon Salmo salar L. Blood parameters were measured and tissue specimens sampled in six groups of fish; one control group (0 minutes), two groups kept in pure seawater in 90 liter tanks for 30 and 120 minutes, two groups treated with the anesthetic isoeugenol for 30 and 120 minutes, and one group kept in pure seawater for 105 minutes and then anaesthetized with metacaine for 15 minutes. RNA quality was assessed with the NanoDrop ND-1000 spectrophotometer (260/280 and 260/230 nm ratios) and with the Agilent Bioanalyzer (28S/18S ratio and RIN data) in samples either preserved in liquefied nitrogen (N2) or in RNAlater. In addition, the transcriptional levels of two fast-responding genes were quantified in gill and brain tissues. Results The results show that physiological stress during sampling does not affect the quality of RNA extracted from fish specimens. However, prolonged sedation (2 hours) resulted in a metabolic alkalosis that again affected the transcriptional levels of genes involved in ionoregulation and respiration. In gills, Na+-K+-ATPase α1b was significantly downregulated and hypoxia inducible factor 1 (HIF1) significantly upregulated after two hours of treatment with isoeugenol, suggesting that this commonly used sedative affects osmo-regulation and respiration in the fish. The results also suggest that for tissue preservation in general it is better to flash-freeze fish specimens in liquefied N2 than to use RNAlater. Conclusion Prolonged sedation may affect the transcription of fast-responding genes in tissues of fish. Two hours of sedation with isoeugenol resulted in downregulation of the Na+-K+-ATPase α1b gene and upregulation of the HIF1 gene in gills of Atlantic salmon. The quality of RNA extracted from tissue specimens, however, was not affected by sedation treatment. Flash-freezing of tissue specimens seems to be the preferred preservation technique, when sampling fish tissue specimens for RNA extraction. PMID:17559653

Comprehensive Assessments of RNA-seq by the SEQC Consortium: FDA-Led Efforts Advance Precision Medicine.

PubMed

Xu, Joshua; Gong, Binsheng; Wu, Leihong; Thakkar, Shraddha; Hong, Huixiao; Tong, Weida

2016-03-15

Studies on gene expression in response to therapy have led to the discovery of pharmacogenomics biomarkers and advances in precision medicine. Whole transcriptome sequencing (RNA-seq) is an emerging tool for profiling gene expression and has received wide adoption in the biomedical research community. However, its value in regulatory decision making requires rigorous assessment and consensus between various stakeholders, including the research community, regulatory agencies, and industry. The FDA-led SEquencing Quality Control (SEQC) consortium has made considerable progress in this direction, and is the subject of this review. Specifically, three RNA-seq platforms (Illumina HiSeq, Life Technologies SOLiD, and Roche 454) were extensively evaluated at multiple sites to assess cross-site and cross-platform reproducibility. The results demonstrated that relative gene expression measurements were consistently comparable across labs and platforms, but not so for the measurement of absolute expression levels. As part of the quality evaluation several studies were included to evaluate the utility of RNA-seq in clinical settings and safety assessment. The neuroblastoma study profiled tumor samples from 498 pediatric neuroblastoma patients by both microarray and RNA-seq. RNA-seq offers more utilities than microarray in determining the transcriptomic characteristics of cancer. However, RNA-seq and microarray-based models were comparable in clinical endpoint prediction, even when including additional features unique to RNA-seq beyond gene expression. The toxicogenomics study compared microarray and RNA-seq profiles of the liver samples from rats exposed to 27 different chemicals representing multiple toxicity modes of action. Cross-platform concordance was dependent on chemical treatment and transcript abundance. Though both RNA-seq and microarray are suitable for developing gene expression based predictive models with comparable prediction performance, RNA-seq offers advantages over microarray in profiling genes with low expression. The rat BodyMap study provided a comprehensive rat transcriptomic body map by performing RNA-Seq on 320 samples from 11 organs in either sex of juvenile, adolescent, adult and aged Fischer 344 rats. Lastly, the transferability study demonstrated that signature genes of predictive models are reciprocally transferable between microarray and RNA-seq data for model development using a comprehensive approach with two large clinical data sets. This result suggests continued usefulness of legacy microarray data in the coming RNA-seq era. In conclusion, the SEQC project enhances our understanding of RNA-seq and provides valuable guidelines for RNA-seq based clinical application and safety evaluation to advance precision medicine.

Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.

PubMed

Mullin, Anne E; Soukatcheva, Galina; Verchere, C Bruce; Chantler, Janet K

2006-05-01

A technique to isolate high-quality intact RNA from murine pancreas is described. This technique involves in situ ductal perfusion of the pancreas with an RNase inhibitor prior to removal of the organ for RNA extraction. In this way, the pancreatic RNases are inhibited in situ allowing good yields of intact RNA, suitable for studies on pancreatic gene transcription by real-time PCR or microarray analysis, to be obtained in a reliable way.

Upf1 senses 3′UTR length to potentiate mRNA decay

PubMed Central

Hogg, J. Robert; Goff, Stephen P.

2010-01-01

Summary The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay. PMID:21029861

Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing.

PubMed

Tourlousse, Dieter M; Yoshiike, Satowa; Ohashi, Akiko; Matsukura, Satoko; Noda, Naohiro; Sekiguchi, Yuji

2017-02-28

High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Feasibility of implementing molecular-guided therapy for the treatment of patients with relapsed or refractory neuroblastoma

PubMed Central

Saulnier Sholler, Giselle L; Bond, Jeffrey P; Bergendahl, Genevieve; Dutta, Akshita; Dragon, Julie; Neville, Kathleen; Ferguson, William; Roberts, William; Eslin, Don; Kraveka, Jacqueline; Kaplan, Joel; Mitchell, Deanna; Parikh, Nehal; Merchant, Melinda; Ashikaga, Takamaru; Hanna, Gina; Lescault, Pamela Jean; Siniard, Ashley; Corneveaux, Jason; Huentelman, Matthew; Trent, Jeffrey

2015-01-01

The primary objective of the study was to evaluate the feasibility and safety of a process which would utilize genome-wide expression data from tumor biopsies to support individualized treatment decisions. Current treatment options for recurrent neuroblastoma are limited and ineffective, with a survival rate of <10%. Molecular profiling may provide data which will enable the practitioner to select the most appropriate therapeutic option for individual patients, thus improving outcomes. Sixteen patients with neuroblastoma were enrolled of which fourteen were eligible for this study. Feasibility was defined as completion of tumor biopsy, pathological evaluation, RNA quality control, gene expression profiling, bioinformatics analysis, generation of a drug prediction report, molecular tumor board yielding a treatment plan, independent medical monitor review, and treatment initiation within a 21 day period. All eligible biopsies passed histopathology and RNA quality control. Expression profiling by microarray and RNA sequencing were mutually validated. The average time from biopsy to report generation was 5.9 days and from biopsy to initiation of treatment was 12.4 days. No serious adverse events were observed and all adverse events were expected. Clinical benefit was seen in 64% of patients as stabilization of disease for at least one cycle of therapy or partial response. The overall response rate was 7% and the progression free survival was 59 days. This study demonstrates the feasibility and safety of performing real-time genomic profiling to guide treatment decision making for pediatric neuroblastoma patients. PMID:25720842

The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data

PubMed Central

Korenkova, Vlasta; Slyskova, Jana; Novosadova, Vendula; Pizzamiglio, Sara; Langerova, Lucie; Bjorkman, Jens; Vycital, Ondrej; Liska, Vaclav; Levy, Miroslav; Veskrna, Karel; Vodicka, Pavel; Vodickova, Ludmila; Kubista, Mikael; Verderio, Paolo

2016-01-01

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing. PMID:27383461

Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops.

PubMed

Christou, Anastasis; Georgiadou, Egli C; Filippou, Panagiota; Manganaris, George A; Fotopoulos, Vasileios

2014-03-01

Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops. Copyright © 2013 Elsevier B.V. All rights reserved.

Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.

PubMed

Butler, Alexandra E; Matveyenko, Aleksey V; Kirakossian, David; Park, Johanna; Gurlo, Tatyana; Butler, Peter C

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6-7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

Presence, infectivity, and stability of enteric viruses in seawater: Relationship to marine water quality in the Florida Keys

USGS Publications Warehouse

Wetz, J.J.; Lipp, E.K.; Griffin, Dale W.; Lukasik, J.; Wait, D.; Sobsey, M.D.; Scott, T.M.; Rose, J.B.

2004-01-01

Concerns about the presence of enteric viruses in the surface waters of the Florida Keys prompted analyses of virus stability and persistence in these waters. In an in vitro study we evaluated the survival of poliovirus and stability of viral RNA in filtered natural seawater (FSW), unfiltered natural seawater (USW), artificial seawater (ASW) and DI water. This study compared cell culture infectivity with direct reverse transcription-polymerase chain reaction analysis. Attenuated poliovirus was seeded in the above water types and incubated in the dark at 22 and 30??C for 60 days. At 22??C, enhanced poliovirus survival and enhanced detection of viral RNA was observed in the seeded DI water control, artificial seawater and FSW samples. Detection of viruses in unfiltered seawater decreased rapidly at both temperatures by both methods of detection, suggesting that in the natural environment detection of enteroviral RNA may indicate a recent contamination event. In addition, in situ sampling in the Florida Keys during the late winter of 2000 revealed the presence of infectious enteroviruses at two sites and no sites exceeded recommended levels of microbial water quality indicators (enterococci or fecal coliform bacteria). ?? 2003 Elsevier Ltd. All rights reserved.

Evaluation of RNA from human trabecular bone and identification of stable reference genes.

PubMed

Cepollaro, Simona; Della Bella, Elena; de Biase, Dario; Visani, Michela; Fini, Milena

2018-06-01

The isolation of good quality RNA from tissues is an essential prerequisite for gene expression analysis to study pathophysiological processes. This study evaluated the RNA isolated from human trabecular bone and defined a set of stable reference genes. After pulverization, RNA was extracted with a phenol/chloroform method and then purified using silica columns. The A260/280 ratio, A260/230 ratio, RIN, and ribosomal ratio were measured to evaluate RNA quality and integrity. Moreover, the expression of six candidates was analyzed by qPCR and different algorithms were applied to assess reference gene stability. A good purity and quality of RNA was achieved according to A260/280 and A260/230 ratios, and RIN values. TBP, YWHAZ, and PGK1 were the most stable reference genes that should be used for gene expression analysis. In summary, the method proposed is suitable for gene expression evaluation in human bone and a set of reliable reference genes has been identified. © 2017 Wiley Periodicals, Inc.

Effect of multiple cycles of freeze-thawing on the RNA quality of lung cancer tissues.

PubMed

Yu, Keke; Xing, Jie; Zhang, Jie; Zhao, Ruiying; Zhang, Ye; Zhao, Lanxiang

2017-09-01

RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyoung Mi; Cho, Hana; Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr

Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinasemore » inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.« less

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

PubMed Central

Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko; Nitharwal, Ram G.; Takase, Ryuichi; Han, Kyu Young; Leslie, Benjamin J.; Liu, Cuiping; Gamper, Howard; Ha, Taekjip; Sanyal, Suparna

2017-01-01

Abstract Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering. PMID:27956502

A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses.

PubMed

Yang, Fan; Wang, Guoping; Xu, Wenxing; Hong, Ni

2017-09-01

Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of Quyu Jiedu granule on microenvironment of ova in patients with endometriosis.

PubMed

Lian, Fang; Li, Xin-Ling; Sun, Zhen-Gao; Zhang, Jian-Wei; Liu, Yan-He; Ma, Feng-Mei

2009-02-01

To observe the effect of Quyu Jiedu Granules (, QJG) on the micro- microenvironment of ova in patients with endometriosis (EM). environment Twenty EM patients who received in vitro fertilization and embryo transfer (IVF-ET) were randomized equally into a treated group and a control group. Further, 20 patients who received IVF-ET due to oviduct factors were enrolled into a non-endometriosis group. The dosage of gonadotrophic hormone used, the number of ova attained, fertilization rate and clinical pregnancy rate were all observed, and the levels of tumor necrosis factor alpha (TNF-right harpoon over left harpoon) and interleukin 6 (IL-6) in follicular fluid as well as their mRNA expressions in ovarian granular cells were detected by RT-PCR on the very day of ovum attainment. The ova attainment (13.80+/-6.87) and fertilization rate (0.69+/-0.31) in the treated group were all higher than the corresponding values in the control group (9.80+/-5.32 and 0.47+/-0.22); the follicular fluid contents of TNF-alpha and IL-6 in the treated group were 1.38+/-0.21 ng/mL and 130.56+/-12.81 pg/mL, respectively, which were lower than those in the control group (1.98+/-0.34 ng/mL and 146.83+/-17.65 pg/mL, respectively). Further, the treated group showed much lower mRNA expressions of TNF-alpha and IL-6 in ovarian granular cells. The elevation of TNF-alpha and IL-6 contents in follicular fluid and their mRNA expressions in ovarian granular cells are possibly related to the low quality of ova in EM; QJG might raise the ova quality by reducing TNF-alpha and IL-6 levels to improve the living micro-environment for the ova.

Global Reprogramming of Host SUMOylation during Influenza Virus Infection

PubMed Central

Domingues, Patricia; Golebiowski, Filip; Tatham, Michael H.; Lopes, Antonio M.; Taggart, Aislynn; Hay, Ronald T.; Hale, Benjamin G.

2015-01-01

Summary Dynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damage, and DNA virus infection. Here, we describe a non-canonical host SUMOylation response to the nuclear-replicating RNA pathogen, influenza virus, and identify viral RNA polymerase activity as a major contributor to SUMO proteome remodeling. Using quantitative proteomics to compare stress-induced SUMOylation responses, we reveal that influenza virus infection triggers unique re-targeting of SUMO to 63 host proteins involved in transcription, mRNA processing, RNA quality control, and DNA damage repair. This is paralleled by widespread host deSUMOylation. Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes. Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression. Our global characterization of influenza virus-triggered SUMO redistribution provides a proteomic resource to understand host nuclear SUMOylation responses to infection. PMID:26549460

HALO--a Java framework for precise transcript half-life determination.

PubMed

Friedel, Caroline C; Kaufmann, Stefanie; Dölken, Lars; Zimmer, Ralf

2010-05-01

Recent improvements in experimental technologies now allow measurements of de novo transcription and/or RNA decay at whole transcriptome level and determination of precise transcript half-lives. Such transcript half-lives provide important insights into the regulation of biological processes and the relative contributions of RNA decay and de novo transcription to differential gene expression. In this article, we present HALO (Half-life Organizer), the first software for the precise determination of transcript half-lives from measurements of RNA de novo transcription or decay determined with microarrays or RNA-seq. In addition, methods for quality control, filtering and normalization are supplied. HALO provides a graphical user interface, command-line tools and a well-documented Java application programming interface (API). Thus, it can be used both by biologists to determine transcript half-lives fast and reliably with the provided user interfaces as well as software developers integrating transcript half-life analysis into other gene expression profiling pipelines. Source code, executables and documentation are available at http://www.bio.ifi.lmu.de/software/halo.

Editing activity for eliminating mischarged tRNAs is essential in mammalian mitochondria

PubMed Central

Hilander, Taru; Zhou, Xiao-Long; Konovalova, Svetlana; Zhang, Fu-Ping; Euro, Liliya; Chilov, Dmitri; Poutanen, Matti; Chihade, Joseph

2018-01-01

Abstract Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms. PMID:29228266

Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

PubMed

Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

2016-08-26

The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.

PubMed

Galichon, P; Amrouche, L; Hertig, A; Brocheriou, I; Rabant, M; Xu-Dubois, Y-C; Ouali, N; Dahan, K; Morin, L; Terzi, F; Rondeau, E; Anglicheau, D

2016-10-01

Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

A Multidisciplinary Biospecimen Bank of Renal Cell Carcinomas Compatible with Discovery Platforms at Mayo Clinic, Scottsdale, Arizona

PubMed Central

Ho, Thai H.; Nateras, Rafael Nunez; Yan, Huihuang; Park, Jin G.; Jensen, Sally; Borges, Chad; Lee, Jeong Heon; Champion, Mia D.; Tibes, Raoul; Bryce, Alan H.; Carballido, Estrella M.; Todd, Mark A.; Joseph, Richard W.; Wong, William W.; Parker, Alexander S.; Stanton, Melissa L.; Castle, Erik P.

2015-01-01

To address the need to study frozen clinical specimens using next-generation RNA, DNA, chromatin immunoprecipitation (ChIP) sequencing and protein analyses, we developed a biobank work flow to prospectively collect biospecimens from patients with renal cell carcinoma (RCC). We describe our standard operating procedures and work flow to annotate pathologic results and clinical outcomes. We report quality control outcomes and nucleic acid yields of our RCC submissions (N=16) to The Cancer Genome Atlas (TCGA) project, as well as newer discovery platforms, by describing mass spectrometry analysis of albumin oxidation in plasma and 6 ChIP sequencing libraries generated from nephrectomy specimens after histone H3 lysine 36 trimethylation (H3K36me3) immunoprecipitation. From June 1, 2010, through January 1, 2013, we enrolled 328 patients with RCC. Our mean (SD) TCGA RNA integrity numbers (RINs) were 8.1 (0.8) for papillary RCC, with a 12.5% overall rate of sample disqualification for RIN <7. Banked plasma had significantly less albumin oxidation (by mass spectrometry analysis) than plasma kept at 25°C (P<.001). For ChIP sequencing, the FastQC score for average read quality was at least 30 for 91% to 95% of paired-end reads. In parallel, we analyzed frozen tissue by RNA sequencing; after genome alignment, only 0.2% to 0.4% of total reads failed the default quality check steps of Bowtie2, which was comparable to the disqualification ratio (0.1%) of the 786-O RCC cell line that was prepared under optimal RNA isolation conditions. The overall correlation coefficients for gene expression between Mayo Clinic vs TCGA tissues ranged from 0.75 to 0.82. These data support the generation of high-quality nucleic acids for genomic analyses from banked RCC. Importantly, the protocol does not interfere with routine clinical care. Collections over defined time points during disease treatment further enhance collaborative efforts to integrate genomic information with outcomes. PMID:26181416

Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.

PubMed

Kresse, Stine H; Namløs, Heidi M; Lorenz, Susanne; Berner, Jeanne-Marie; Myklebost, Ola; Bjerkehagen, Bodil; Meza-Zepeda, Leonardo A

2018-01-01

Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.

Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates*

PubMed Central

Tennstaedt, Annette; Pöpsel, Simon; Truebestein, Linda; Hauske, Patrick; Brockmann, Anke; Schmidt, Nina; Irle, Inga; Sacca, Barbara; Niemeyer, Christof M.; Brandt, Roland; Ksiezak-Reding, Hanna; Tirniceriu, Anca Laura; Egensperger, Rupert; Baldi, Alfonso; Dehmelt, Leif; Kaiser, Markus; Huber, Robert; Clausen, Tim; Ehrmann, Michael

2012-01-01

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. PMID:22535953

Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis

PubMed Central

Wang, Cynthia Xin-Zhao; Zhang, Kaiyan; Aredo, Bogale; Lu, Hua; Ufret-Vincenty, Rafael L.

2012-01-01

RPE cells are involved in the pathogenesis of many retinal diseases. Accurate analysis of RPE gene expression profiles in different scenarios will increase our understanding of disease mechanisms. Our objective in this study was to develop an improved method for the isolation of RPE cells, specifically for RNA analysis. Mouse RPE cells were isolated using different techniques, including mechanical dissociation techniques and a new technique we refer to here as “Simultaneous RPE cell Isolation and RNA Stabilization” (SRIRS method). RNA was extracted from the RPE cells. An RNA bioanalyzer was used to determine the quantity and quality of RNA. qPCR was used to determine contamination with non-RPE-derived RNA. Several parameters with a potential impact on the isolation protocol were studied and optimized. A marked improvement in the quantity and quality of RPE-derived RNA was obtained with the SRIRS technique. We could get the RPE in direct contact with the RNA protecting agent within 1 minute of enucleation, and the RPE isolated within 11 minutes of enucleation. There was no significant contamination with vascular, choroidal or scleral-derived RNA. We have developed a fast, easy and reliable method for the isolation of RPE cells that leads to a high yield of RPE-derived RNA while preserving its quality. We believe this technique will be useful for future studies looking at gene expression profiles of RPE cells and their role in the pathophysiology of retinal diseases. PMID:22721721

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

PubMed

Lalev, A I; Abeyrathne, P D; Nazar, R N

2000-09-08

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. Copyright 2000 Academic Press.

Feasibility of the salivary transcriptome as a novel biomarker in determining disease susceptibility.

PubMed

Hidayat, M F H; Milne, T; Cullinan, M P; Seymour, G J

2018-06-01

The salivary transcriptome may present as a readily available and non-invasive source of potential biomarkers. The development of chronic periodontitis is determined by individual patient susceptibility; hence, the aim of this study was to determine the potential of the salivary transcriptome as a biomarker of disease susceptibility using chronic periodontitis as an example. Using an Oragene ® RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction. Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92 μg/500 μL to 62.85 μg/500 μL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21 ± 12.71 μg/500 μL for the periodontitis group and 15.97 ± 23.47 μg/500 μL for the control group. Further the RNA purity (based on the A 260 /A 280 ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0 ± 0.1). The study samples, showed 2 distinct bands at 23S (3800 bp) and 16S (1500 bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA. This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

PubMed Central

Ruocco, Nadia; Costantini, Susan; Zupo, Valerio; Romano, Giovanna; Ianora, Adrianna; Fontana, Angelo; Costantini, Maria

2017-01-01

The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism. PMID:28199408

De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies.

PubMed

Mitsuhashi, Kana; Ito, Daisuke; Mashima, Kyoko; Oyama, Munenori; Takahashi, Shinichi; Suzuki, Norihiro

2017-12-04

Aberrant RNA-binding proteins form the core of the neurodegeneration cascade in spectrums of disease, such as amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn)RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients and those share several common features, including harboring an aggregation-prone prion-like domain (PrLD) containing a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence and rich in glutamine and/or asparagine. Additinally, these six molecules are components of RNA granules involved in RNA quality control and become mislocated from the nucleus to form cytoplasmic inclusion bodies (IBs) in the ALS/FTD-affected brain. To reveal the essential mechanisms involved in ALS/FTD-related cytotoxicity associated with RNA-binding proteins containing PrLDs, we designed artificial RNA-binding proteins harboring G/S-Y-G/S-motif repeats with and without enriched glutamine residues and nuclear-import/export-signal sequences and examined their cytotoxicity in vitro. These proteins recapitulated features of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and components of RNA granules, and cytotoxicity instigation. These findings indicated that these artificial RNA-binding proteins mimicked features of ALS-linked molecules and allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis.

RNA isolation from mouse pancreas: a ribonuclease-rich tissue.

PubMed

Azevedo-Pouly, Ana Clara P; Elgamal, Ola A; Schmittgen, Thomas D

2014-08-02

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

The effects of Bacillus coagulans-fermented and non-fermented Ginkgo biloba on abdominal fat deposition and meat quality of Peking duck.

PubMed

Liu, Xiaoyan; Cao, Guanjun; Zhou, Jinglong; Yao, Xuan; Fang, Binghu

2017-07-01

In order to evaluate the effects of Bacillus coagulans-fermented Ginkgo biloba (FG) and non-fermented G. biloba (NFG) on abdominal fat deposition and meat quality, 270 female Peking ducks were randomly assigned to the following experimental groups: a control group (fed a basal diet), an NFG group (fed a basal diet + 0.3% NFG), and an FG group (fed a basal diet + 0.3% FG). Body weight and feed intake were recorded weekly, and feed conversion ratio was calculated to assess growth performance. After 6 wk, 18 ducks from each group were killed. Abdominal fat ratio and pH (at 45 min and 24 h postmortem), color parameters (lightness, redness, and yellowness), water-holding capacity, cooking loss, shear force, and intramuscular fat and fatty acid contents were measured. Six more ducks were killed to isolate RNA from their abdominal fat tissue for measurements of peroxisome proliferator-activated receptor-γ (PPARγ), obese (leptin), and adiponectin (ADP) expression using real-time polymerase chain reaction. The results revealed that body weight gain was higher in the FG group than in the control and NFG groups, whereas feed conversion ratio was lower (P < 0.05). The abdominal fat contents were lower in the NFG and FG groups than in the control group (P < 0.05). The NFG and FG groups had lower levels of saturated fatty acids (mainly palmitic acid) and higher levels of polyunsaturated fatty acids (mainly linoleic acid and arachidonic acid) than the control group. The mRNA expressions of PPARγ, leptin, and ADP in abdominal fat tissue were significantly increased in the NFG and FG groups, and the mRNA expression of PPARγ was higher in the FG group than in the NFG group (P < 0.05). These results suggest that fermenting G. biloba reduces the deposition of abdominal fat and improves the fatty acid profile of Peking duck meat. © 2017 Poultry Science Association Inc.

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals

PubMed Central

Machado, Ana Karla; Brown, Neil A; Urban, Martin; Kanyuka, Kostya

2017-01-01

Abstract Fusarium graminearum is a major fungal pathogen of cereals worldwide, causing seedling, stem base and floral diseases, including Fusarium head blight (FHB). In addition to yield and quality losses, FHB contaminates cereal grain with mycotoxins, including deoxynivalenol, which are harmful to human, animal and ecosystem health. Currently, FHB control is only partially effective due to several intractable problems. RNA interference (RNAi) is a natural mechanism that regulates gene expression. RNAi has been exploited in the development of new genomic tools that allow the targeted silencing of genes of interest in many eukaryotes. Host‐induced gene silencing (HIGS) is a transgenic technology used to silence fungal genes in planta during attempted infection and thereby reduces disease levels. HIGS relies on the host plant's ability to produce mobile small interfering RNA molecules, generated from long double‐stranded RNA, which are complementary to targeted fungal genes. These molecules are transferred from the plant to invading fungi via an uncharacterised mechanism, to cause gene silencing. Here, we describe recent advances in RNAi‐mediated control of plant pathogenic fungi, highlighting the key advantages and disadvantages. We then discuss the developments and implications of combining HIGS with other methods of disease control. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:28967180

Factors That Influence the Quality of RNA From the Pancreas of Organ Donors

PubMed Central

Philips, Tiffany; Kusmartseva, Irina; Gerling, Ivan C.; Campbell-Thompson, Martha; Wasserfall, Clive; Pugliese, Alberto; Longmate, Jeffrey A.; Schatz, Desmond A.; Atkinson, Mark A.; Kaddis, John S.

2016-01-01

Objectives Attaining high quality RNA from the tissues or organs of deceased donors used for research can be challenging due to physiological and logistical considerations. In this investigation, Methods RNA Integrity Number (RIN) was determined in pancreatic samples from 236 organ donors and utilized to define high (≥6.5) and low (≤4.5) quality RNA. Logistic regression was used to evaluate the potential effects of novel or established organ and donor factors on RIN. Results Univariate analysis revealed donor cause of death (Odds Ratio [OR]=0.35, 95% Confidence Interval [CI]=0.15–0.77, p=0.01), prolonged tissue storage prior to RNA extraction (OR=0.65, 95%CI 0.52–0.79, p<0.01), pancreas region sampled (multiple comparisons, p<0.01), and sample type (OR=0.32, 95%CI 0.15–0.67, p<0.01) negatively influenced outcome. Conversely, duration of final hospitalization (OR=3.95, 95%CI 1.59–10.37, p<0.01) and sample collection protocol (OR=8.48, 95%CI 3.96–19.30, p<0.01) positively impacted outcome. Islet RNA obtained via laser capture microdissection improved RIN when compared to total pancreatic RNA from the same donor (∆RIN=1.3, 95%CI 0.6–2.0, p<0.01). Conclusions A multivariable model demonstrates that autopsy- and biopsy-free human pancreata received, processed, and preserved at a single center, using optimized procedures, from organ donors dying of anoxia with normal lipase levels increase the odds of obtaining high-quality RNA. PMID:27984510

Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.).

PubMed

Rodrigues, Simone M; Soares, Virgínia L F; de Oliveira, Tahise M; Gesteira, Abelmon S; Otoni, Wagner C; Costa, Marcio G C

2007-11-01

The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.

Genome-wide assessment of differential translations with ribosome profiling data.

PubMed

Xiao, Zhengtao; Zou, Qin; Liu, Yu; Yang, Xuerui

2016-04-04

The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations.

An efficient field and laboratory workflow for plant phylotranscriptomic projects1

PubMed Central

Yang, Ya; Moore, Michael J.; Brockington, Samuel F.; Timoneda, Alfonso; Feng, Tao; Marx, Hannah E.; Walker, Joseph F.; Smith, Stephen A.

2017-01-01

Premise of the study: We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation. Methods and Results: A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae. Conclusions: Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10–12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds. PMID:28337391

A novel method to produce armored double-stranded DNA by encapsulation of MS2 viral capsids.

PubMed

Zhang, Lei; Sun, Yu; Chang, Le; Jia, Tingting; Wang, Guojing; Zhang, Rui; Zhang, Kuo; Li, Jinming

2015-09-01

With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successfully package double-stranded DNA of 1.3-, 3-, 3.5-, and 6.5-kb length into viral capsids with high reassembly efficiency. This is the first application of RNA bacteriophage MS2 as a platform to encapsulate double-stranded DNA, forming virus-like particles (VLPs) which were indistinguishable from native MS2 capsids in size and morphology. Moreover, by analyzing the interaction mechanism of pac site and the MS2 coat protein (CP), we found that in addition to the recognized initiation signal TR-RNA, TR-DNA can also trigger spontaneous reassembly of CP dimers, providing a more convenient and feasible method of assembly. In conclusion, this straightforward and reliable manufacturing approach makes armored DNA an ideal control and standard for use in clinical laboratory tests and diagnostics, possessing prospects for broad application, especially providing a new platform for the production of quality controls for DNA viruses.

Rewiring protein synthesis: From natural to synthetic amino acids.

PubMed

Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

2017-11-01

The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Emerging functions of alternative splicing coupled with nonsense-mediated decay.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-08-01

Higher eukaryotes rely on AS (alternative splicing) of pre-mRNAs (mRNA precursors) to generate more than one protein product from a single gene and to regulate mRNA stability and translational activity. An important example of the latter function involves an interplay between AS and NMD (nonsense-mediated decay), a cytoplasmic quality control mechanism eliminating mRNAs containing PTCs (premature translation termination codons). Although originally identified as an error surveillance process, AS-NMD additionally provides an efficient strategy for deterministic regulation of gene expression outputs. In this review, we discuss recently published examples of AS-NMD and delineate functional contexts where recurrent use of this mechanism orchestrates expression of important genes.

Synthetic Spike-in Standards Improve Run-Specific Systematic Error Analysis for DNA and RNA Sequencing

PubMed Central

Zook, Justin M.; Samarov, Daniel; McDaniel, Jennifer; Sen, Shurjo K.; Salit, Marc

2012-01-01

While the importance of random sequencing errors decreases at higher DNA or RNA sequencing depths, systematic sequencing errors (SSEs) dominate at high sequencing depths and can be difficult to distinguish from biological variants. These SSEs can cause base quality scores to underestimate the probability of error at certain genomic positions, resulting in false positive variant calls, particularly in mixtures such as samples with RNA editing, tumors, circulating tumor cells, bacteria, mitochondrial heteroplasmy, or pooled DNA. Most algorithms proposed for correction of SSEs require a data set used to calculate association of SSEs with various features in the reads and sequence context. This data set is typically either from a part of the data set being “recalibrated” (Genome Analysis ToolKit, or GATK) or from a separate data set with special characteristics (SysCall). Here, we combine the advantages of these approaches by adding synthetic RNA spike-in standards to human RNA, and use GATK to recalibrate base quality scores with reads mapped to the spike-in standards. Compared to conventional GATK recalibration that uses reads mapped to the genome, spike-ins improve the accuracy of Illumina base quality scores by a mean of 5 Phred-scaled quality score units, and by as much as 13 units at CpG sites. In addition, since the spike-in data used for recalibration are independent of the genome being sequenced, our method allows run-specific recalibration even for the many species without a comprehensive and accurate SNP database. We also use GATK with the spike-in standards to demonstrate that the Illumina RNA sequencing runs overestimate quality scores for AC, CC, GC, GG, and TC dinucleotides, while SOLiD has less dinucleotide SSEs but more SSEs for certain cycles. We conclude that using these DNA and RNA spike-in standards with GATK improves base quality score recalibration. PMID:22859977

UrQt: an efficient software for the Unsupervised Quality trimming of NGS data.

PubMed

Modolo, Laurent; Lerat, Emmanuelle

2015-04-29

Quality control is a necessary step of any Next Generation Sequencing analysis. Although customary, this step still requires manual interventions to empirically choose tuning parameters according to various quality statistics. Moreover, current quality control procedures that provide a "good quality" data set, are not optimal and discard many informative nucleotides. To address these drawbacks, we present a new quality control method, implemented in UrQt software, for Unsupervised Quality trimming of Next Generation Sequencing reads. Our trimming procedure relies on a well-defined probabilistic framework to detect the best segmentation between two segments of unreliable nucleotides, framing a segment of informative nucleotides. Our software only requires one user-friendly parameter to define the minimal quality threshold (phred score) to consider a nucleotide to be informative, which is independent of both the experiment and the quality of the data. This procedure is implemented in C++ in an efficient and parallelized software with a low memory footprint. We tested the performances of UrQt compared to the best-known trimming programs, on seven RNA and DNA sequencing experiments and demonstrated its optimality in the resulting tradeoff between the number of trimmed nucleotides and the quality objective. By finding the best segmentation to delimit a segment of good quality nucleotides, UrQt greatly increases the number of reads and of nucleotides that can be retained for a given quality objective. UrQt source files, binary executables for different operating systems and documentation are freely available (under the GPLv3) at the following address: https://lbbe.univ-lyon1.fr/-UrQt-.html .

Multigen-2 Pre-Flight Testing: Science Testing Unit (STU) and Stowage Conditions

NASA Astrophysics Data System (ADS)

Kittang, A.-I.; Kvaloy, B.; Berg, C.; Rakvaag, G.; Iversen, T.-H.

2008-06-01

The Multigen-2 experiment Science Testing Unit (STU) proved to be a useful tool in optimizing experiment environment settings for cultivation of Arabidopsis thaliana (Col-0) in the European Modular Cultivation System (EMCS). By using the EMCS Experiment Reference Model (ERM); light, temperature and air flow regimes for optimal growth could be tested. Healthy seedlings were obtained using the STU#2 and STU#3 in the EMCS ERM. It was concluded that the Experiment Container Development Kit (ECDK) is unsuitable for the Multigen-2 testing due to limitation in the ECDK temperature control. The results from the stowage condition tests showed that the selected growth medium (agar) can be used after 3 months at +4°C. The seeds show a germination rate of ≥80% after sterilisation and stowed for 5 months. The Multigen-2 plant samples will be fixed in RNA later and stored at - 80 °C. Three methods with different RNA isolation kits showed that the Qiagen kit (#74904) gave the highest amount and the best quality of Total RNA from RNA Later and frozen samples. The amount of plant material from one cultivation chamber gives two RNA isolations. Each of the isolations gives Total RNA sufficient for at least two microarray analyses.

External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA

PubMed Central

Jia, Tingting; Zhang, Lei; Wang, Guojing; Zhang, Rui; Zhang, Kuo; Lin, Guigao; Xie, Jiehong; Wang, Lunan; Li, Jinming

2015-01-01

In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7%) were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL). In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection of measles virus. PMID:26244795

External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA.

PubMed

Zhang, Dong; Sun, Yu; Jia, Tingting; Zhang, Lei; Wang, Guojing; Zhang, Rui; Zhang, Kuo; Lin, Guigao; Xie, Jiehong; Wang, Lunan; Li, Jinming

2015-01-01

In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7%) were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL). In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection of measles virus.

Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds

PubMed Central

2013-01-01

Background High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants. Findings Here we report the use of adsorption property of activated charcoal (0.03%–0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number. Conclusions The RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds. PMID:23537338

Cytosolic Hsp70 and co-chaperones constitute a novel system for tRNA import into the nucleus

PubMed Central

Takano, Akira; Kajita, Takuya; Mochizuki, Makoto; Endo, Toshiya; Yoshihisa, Tohru

2015-01-01

tRNAs are unique among various RNAs in that they shuttle between the nucleus and the cytoplasm, and their localization is regulated by nutrient conditions. Although nuclear export of tRNAs has been well documented, the import machinery is poorly understood. Here, we identified Ssa2p, a major cytoplasmic Hsp70 in Saccharomyces cerevisiae, as a tRNA-binding protein whose deletion compromises nuclear accumulation of tRNAs upon nutrient starvation. Ssa2p recognizes several structural features of tRNAs through its nucleotide-binding domain, but prefers loosely-folded tRNAs, suggesting that Ssa2p has a chaperone-like activity for RNAs. Ssa2p also binds Nup116, one of the yeast nucleoporins. Sis1p and Ydj1p, cytoplasmic co-chaperones for Ssa proteins, were also found to contribute to the tRNA import. These results unveil a novel function of the Ssa2p system as a tRNA carrier for nuclear import by a novel mode of substrate recognition. Such Ssa2p-mediated tRNA import likely contributes to quality control of cytosolic tRNAs. DOI: http://dx.doi.org/10.7554/eLife.04659.001 PMID:25853343

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo

PubMed Central

Coles, Andrew H.; Osborn, Maire F.; Alterman, Julia F.; Turanov, Anton A.; Godinho, Bruno M.D.C.; Kennington, Lori; Chase, Kathryn; Aronin, Neil

2016-01-01

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene® branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo. PMID:26595721

Gene expression patterns regulating the seed metabolism in relation to deterioration/ageing of primed mung bean (Vigna radiata L.) seeds.

PubMed

Sharma, Satyendra Nath; Maheshwari, Ankita; Sharma, Chitra; Shukla, Nidhi

2018-03-01

We are proposing mechanisms to account for the loss of viability (seed deterioration/ageing) and enhancement in seed quality (post-storage priming treatment). In order to understand the regulatory mechanism of these traits, we conducted controlled deterioration (CD) test for up to 8 d using primed mung bean seeds and examined how CD effects the expression of many genes, regulating the seed metabolism in relation to CD and priming. Germination declined progressively with increased duration of CD, and the priming treatment completely/partially reversed the inhibition depending on the duration of CD. The loss of germination capacity by CD was accompanied by a reduction in total RNA content and RNA integrity, indicating that RNA quantity and quality impacts seed longevity. Expression analysis revealed that biosynthesis genes of GA, ethylene, ABA and ROS-scavenging enzymes were differentially affected in response to duration of CD and priming, suggesting coordinately regulated mechanisms for controlling the germination capacity of seeds by modifying the permeability characteristics of biological membranes and activities of different enzymes. ABA genes were highly expressed when germination was delayed and inhibited by CD. Whereas, GA and ethylene genes were more highly expressed when germination was enhanced and permitted by priming under similar conditions. GSTI, a well characterized enzyme family involved in stress tolerance, was expressed in primed seeds over the period of CD, suggesting an additional protection against deterioration. The results are discussed in light of understanding the mechanisms underlying longevity/priming which are important issues economically and ecologically. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Zearalenone and 17 β-estradiol induced damages in male rats reproduction potential; evidence for ERα and ERβ receptors expression and steroidogenesis.

PubMed

Adibnia, Elmira; Razi, Mazdak; Malekinejad, Hassan

2016-09-15

The estrogen receptors (ERs)-dependent effects of Zearalenone (ZEA) on structure and function of the testis as well as sperm parameters were compared with 17-β estradiol as endogenous substance. For this purpose, 30 mature male rats were assigned into five groups as; control (appropriate volume of normal saline, i. p.), ZEA-received (1, 2 and 4 mg/kg, b. w., i. p.) and 17 β-estradiol (E2)-received (appropriate dose of 0.1 mg/kg, i. p.). Following 28 days, the mRNA levels of estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) in the testis and sperms and the expression of them at protein levels in testicles were estimated. Mitochondrial content of germinal epithelium, Leydig cells steroid foci, sperm quality parameters and serum level of testosterone were assessed. Fluorescent techniques were used for analyzing apoptosis and mRNA damage in necrotic cells. ZEA reduced the mRNA and protein levels of ERα in testicles while up-regulated the ERβ expression. The mRNA level of ERα decreased in sperms of ZEA and E2-received animals. No remarkable changes were found for ERβ expression in sperms from ZEA and E2-received animals. ZEA reduced the Leydig cells steroidogenesis, mitochondrial content of germinal cells and elevated cellular apoptosis and necrosis dose-dependently. E2 reduced the testosterone concentration, enhanced the apoptosis and reduced sperm quality. Our data suggest that ZEA-induced detrimental effects in the structure and function of testis, may attribute to changing the ERs expression at mRNA and translational level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-wide identification of miRNAs and lncRNAs in Cajanus cajan.

PubMed

Nithin, Chandran; Thomas, Amal; Basak, Jolly; Bahadur, Ranjit Prasad

2017-11-15

Non-coding RNAs (ncRNAs) are important players in the post transcriptional regulation of gene expression (PTGR). On one hand, microRNAs (miRNAs) are an abundant class of small ncRNAs (~22nt long) that negatively regulate gene expression at the levels of messenger RNAs stability and translation inhibition, on the other hand, long ncRNAs (lncRNAs) are a large and diverse class of transcribed non-protein coding RNA molecules (> 200nt) that play both up-regulatory as well as down-regulatory roles at the transcriptional level. Cajanus cajan, a leguminosae pulse crop grown in tropical and subtropical areas of the world, is a source of high value protein to vegetarians or very poor populations globally. Hence, genome-wide identification of miRNAs and lncRNAs in C. cajan is extremely important to understand their role in PTGR with a possible implication to generate improve variety of crops. We have identified 616 mature miRNAs in C. cajan belonging to 118 families, of which 578 are novel and not reported in MirBase21. A total of 1373 target sequences were identified for 180 miRNAs. Of these, 298 targets were characterized at the protein level. Besides, we have also predicted 3919 lncRNAs. Additionally, we have identified 87 of the predicted lncRNAs to be targeted by 66 miRNAs. miRNA and lncRNAs in plants are known to control a variety of traits including yield, quality and stress tolerance. Owing to its agricultural importance and medicinal value, the identified miRNA, lncRNA and their targets in C. cajan may be useful for genome editing to improve better quality crop. A thorough understanding of ncRNA-based cellular regulatory networks will aid in the improvement of C. cajan agricultural traits.

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the Yeast Saccharomyces cerevisiae

PubMed Central

Hopper, Anita K.

2013-01-01

Transfer RNAs (tRNAs) are essential for protein synthesis. In eukaryotes, tRNA biosynthesis employs a specialized RNA polymerase that generates initial transcripts that must be subsequently altered via a multitude of post-transcriptional steps before the tRNAs beome mature molecules that function in protein synthesis. Genetic, genomic, biochemical, and cell biological approaches possible in the powerful Saccharomyces cerevisiae system have led to exciting advances in our understandings of tRNA post-transcriptional processing as well as to novel insights into tRNA turnover and tRNA subcellular dynamics. tRNA processing steps include removal of transcribed leader and trailer sequences, addition of CCA to the 3′ mature sequence and, for tRNAHis, addition of a 5′ G. About 20% of yeast tRNAs are encoded by intron-containing genes. The three-step splicing process to remove the introns surprisingly occurs in the cytoplasm in yeast and each of the splicing enzymes appears to moonlight in functions in addition to tRNA splicing. There are 25 different nucleoside modifications that are added post-transcriptionally, creating tRNAs in which ∼15% of the residues are nucleosides other than A, G, U, or C. These modified nucleosides serve numerous important functions including tRNA discrimination, translation fidelity, and tRNA quality control. Mature tRNAs are very stable, but nevertheless yeast cells possess multiple pathways to degrade inappropriately processed or folded tRNAs. Mature tRNAs are also dynamic in cells, moving from the cytoplasm to the nucleus and back again to the cytoplasm; the mechanism and function of this retrograde process is poorly understood. Here, the state of knowledge for tRNA post-transcriptional processing, turnover, and subcellular dynamics is addressed, highlighting the questions that remain. PMID:23633143

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Implementation of Quality Management in Core Service Laboratories

PubMed Central

Creavalle, T.; Haque, K.; Raley, C.; Subleski, M.; Smith, M.W.; Hicks, B.

2010-01-01

CF-28 The Genetics and Genomics group of the Advanced Technology Program of SAIC-Frederick exists to bring innovative genomic expertise, tools and analysis to NCI and the scientific community. The Sequencing Facility (SF) provides next generation short read (Illumina) sequencing capacity to investigators using a streamlined production approach. The Laboratory of Molecular Technology (LMT) offers a wide range of genomics core services including microarray expression analysis, miRNA analysis, array comparative genome hybridization, long read (Roche) next generation sequencing, quantitative real time PCR, transgenic genotyping, Sanger sequencing, and clinical mutation detection services to investigators from across the NIH. As the technology supporting this genomic research becomes more complex, the need for basic quality processes within all aspects of the core service groups becomes critical. The Quality Management group works alongside members of these labs to establish or improve processes supporting operations control (equipment, reagent and materials management), process improvement (reengineering/optimization, automation, acceptance criteria for new technologies and tech transfer), and quality assurance and customer support (controlled documentation/SOPs, training, service deficiencies and continual improvement efforts). Implementation and expansion of quality programs within unregulated environments demonstrates SAIC-Frederick's dedication to providing the highest quality products and services to the NIH community.

RNA sample preparation applied to gene expression profiling for the horse biological passport.

PubMed

Bailly-Chouriberry, Ludovic; Baudoin, Florent; Cormant, Florence; Glavieux, Yohan; Loup, Benoit; Garcia, Patrice; Popot, Marie-Agnès; Bonnaire, Yves

2017-09-01

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgene® Blood RNA Kit for horse gene expression analysis in blood collected on PAXgene® tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Transcriptomics of mRNA and egg quality in farmed fish: Some recent developments and future directions.

PubMed

Sullivan, Craig V; Chapman, Robert W; Reading, Benjamin J; Anderson, Paul E

2015-09-15

Maternal mRNA transcripts deposited in growing oocytes regulate early development and are under intensive investigation as determinants of egg quality. The research has evolved from single gene studies to microarray and now RNA-Seq analyses in which mRNA expression by virtually every gene can be assessed and related to gamete quality. Such studies have mainly focused on genes changing two- to several-fold in expression between biological states, and have identified scores of candidate genes and a few gene networks whose functioning is related to successful development. However, ever-increasing yields of information from high throughput methods for detecting transcript abundance have far outpaced progress in methods for analyzing the massive quantities of gene expression data, and especially for meaningful relation of whole transcriptome profiles to gamete quality. We have developed a new approach to this problem employing artificial neural networks and supervised machine learning with other novel bioinformatics procedures to discover a previously unknown level of ovarian transcriptome function at which minute changes in expression of a few hundred genes is highly predictive of egg quality. In this paper, we briefly review the progress in transcriptomics of fish egg quality and discuss some future directions for this field of study. Copyright © 2015 Elsevier Inc. All rights reserved.

Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

PubMed

Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

2015-12-29

Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.

Factors That Influence the Quality of RNA From the Pancreas of Organ Donors.

PubMed

Philips, Tiffany; Kusmartseva, Irina; Gerling, Ivan C; Campbell-Thompson, Martha; Wasserfall, Clive; Pugliese, Alberto; Longmate, Jeffrey A; Schatz, Desmond A; Atkinson, Mark A; Kaddis, John S

2017-02-01

Attaining high-quality RNA from the tissues or organs of deceased donors used for research can be challenging due to physiological and logistical considerations. In this investigation, METHODS: RNA Integrity Number (RIN) was determined in pancreatic samples from 236 organ donors and used to define high (≥6.5) and low (≤4.5) quality RNAs. Logistic regression was used to evaluate the potential effects of novel or established organ and donor factors on RIN. Univariate analysis revealed donor cause of death (odds ratio [OR], 0.35; 95% confidence interval [CI], 0.15-0.77; P = 0.01), prolonged tissue storage before RNA extraction (OR, 0.65; 95% CI, 0.52-0.79; P < 0.01), pancreas region sampled (multiple comparisons, P < 0.01), and sample type (OR, 0.32; 95% CI, 0.15-0.67; P < 0.01) negatively influenced outcome. Conversely, duration of final hospitalization (OR, 3.95; 95% CI, 1.59-10.37; P < 0.01) and sample collection protocol (OR, 8.48; 95% CI, 3.96-19.30; P < 0.01) positively impacted outcome. Islet RNA obtained via laser capture microdissection improved RIN when compared with total pancreatic RNA from the same donor (ΔRIN = 1.3; 95% CI, 0.6-2.0; P < 0.01). A multivariable model demonstrates that autopsy-free and biopsy-free human pancreata received, processed, and preserved at a single center, using optimized procedures, from organ donors dying of anoxia with normal lipase levels increase the odds of obtaining high-quality RNA.

Sampling and RNA quality for successful diagnostics using quantitative PCR

USDA-ARS?s Scientific Manuscript database

Diagnostic analyses of RNA targets are widely used in honey bee pathology. These diagnostics can be compromised by the actions of endogenous RNA-degrading enzymes activated upon bee death. RNA degradation can be minimized by storage at ultra-cold temperatures or by immersion in high-salt buffers. H...

Dietary calcium deficiency in laying ducks impairs eggshell quality by suppressing shell biomineralization.

PubMed

Chen, Wei; Zhao, Fei; Tian, Zhi Mei; Zhang, Han Xing; Ruan, Dong; Li, Yan; Wang, Shuang; Zheng, Chun Tian; Lin, Ying Cai

2015-10-01

The objective of this study was to determine the effects of dietary calcium deficiency on the process of shell formation. Four hundred and fifty female ducks (Anas platyrhynchos) at 22 weeks were randomly assigned to three groups. Ducks were fed one of two calcium-deficient diets (containing 1.8% or 0.38% calcium, respectively) or a calcium-adequate control diet (containing 3.6% calcium) for 67 days (depletion period) and then all ducks were fed a calcium-adequate diet for an additional 67 days (repletion period). Compared with the calcium-adequate control, the average shell thickness, egg shell weight, breaking strength, mammillae density and mammillary knob thickness of shell from ducks that consumed the diet with 0.38% calcium were significantly decreased (P<0.05) during the depletion period, accompanied by reduced tibia quality. The mRNA expression of both secreted phosphoprotein 1 (SPP1) and carbonic anhydrase 2 (CA2) in the uterus was decreased after feeding calcium-deficient diets (1.8% or 0.38% calcium). mRNA transcripts of calbindin 1 (CALB1), an important protein responsible for calcium transport, and the matrix protein genes ovocalyxin-32 (OCX-32) and ovocleidin-116 (OC-116) were reduced in ducks fed 0.38% calcium but not 1.8% calcium. Plasma estradiol concentration was decreased by both of the calcium-deficient diets (P<0.05). The impaired shell quality and suppressed functional proteins involved in shell formation could be reversed by repletion of dietary calcium. The results of the present study suggest that dietary calcium deficiency negatively affects eggshell quality and microarchitecture, probably by suppressing shell biomineralization. © 2015. Published by The Company of Biologists Ltd.

Template-Based Modeling of Protein-RNA Interactions.

PubMed

Zheng, Jinfang; Kundrotas, Petras J; Vakser, Ilya A; Liu, Shiyong

2016-09-01

Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes.

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

PubMed

Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

2015-01-01

In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

Controlling nuclear RNA levels.

PubMed

Schmid, Manfred; Jensen, Torben Heick

2018-05-10

RNA turnover is an integral part of cellular RNA homeostasis and gene expression regulation. Whereas the cytoplasmic control of protein-coding mRNA is often the focus of study, we discuss here the less appreciated role of nuclear RNA decay systems in controlling RNA polymerase II (RNAPII)-derived transcripts. Historically, nuclear RNA degradation was found to be essential for the functionalization of transcripts through their proper maturation. Later, it was discovered to also be an important caretaker of nuclear hygiene by removing aberrant and unwanted transcripts. Recent years have now seen a set of new protein complexes handling a variety of new substrates, revealing functions beyond RNA processing and the decay of non-functional transcripts. This includes an active contribution of nuclear RNA metabolism to the overall cellular control of RNA levels, with mechanistic implications during cellular transitions.

Improving the Quality of Positive Datasets for the Establishment of Machine Learning Models for pre-microRNA Detection.

PubMed

Demirci, Müşerref Duygu Saçar; Allmer, Jens

2017-07-28

MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of protein abundance and thus have a great impact on the resulting phenotype. It is, therefore, no wonder that they have been implicated in many diseases ranging from virus infections to cancer. This impact on the phenotype leads to a great interest in establishing the miRNAs of an organism. Experimental methods are complicated which led to the development of computational methods for pre-miRNA detection. Such methods generally employ machine learning to establish models for the discrimination between miRNAs and other sequences. Positive training data for model establishment, for the most part, stems from miRBase, the miRNA registry. The quality of the entries in miRBase has been questioned, though. This unknown quality led to the development of filtering strategies in attempts to produce high quality positive datasets which can lead to a scarcity of positive data. To analyze the quality of filtered data we developed a machine learning model and found it is well able to establish data quality based on intrinsic measures. Additionally, we analyzed which features describing pre-miRNAs could discriminate between low and high quality data. Both models are applicable to data from miRBase and can be used for establishing high quality positive data. This will facilitate the development of better miRNA detection tools which will make the prediction of miRNAs in disease states more accurate. Finally, we applied both models to all miRBase data and provide the list of high quality hairpins.

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

PubMed

Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery. Copyright © 2017 Pereira et al.

Bacterial community changes in copper and PEX drinking water pipeline biofilms under extra disinfection and magnetic water treatment.

PubMed

Inkinen, J; Jayaprakash, B; Ahonen, M; Pitkänen, T; Mäkinen, R; Pursiainen, A; Santo Domingo, J W; Salonen, H; Elk, M; Keinänen-Toivola, M M

2018-02-01

To study the stability of biofilms and water quality in pilot scale drinking water copper and PEX pipes in changing conditions (extra disinfection, magnetic water treatment, MWT). Next-generation sequencing (NGS) of 16S ribosomal RNA genes (rDNA) to describe total bacterial community and ribosomal RNA (rRNA) to describe active bacterial members in addition to traditional microbiological methods were applied. Biofilms from control copper and PEX pipes shared same most abundant bacteria (Methylobacterium spp., Sphingomonas spp., Zymomonas spp.) and average species diversities (Shannon 3·8-4·2) in rDNA and rRNA libraries, whereas few of the taxa differed by their abundance such as lower total Mycobacterium spp. occurrence in copper (<0·02%) to PEX (<0·2%) pipes. Extra disinfection (total chlorine increase from c. 0·5 to 1 mg l -1 ) affected total and active population in biofilms seen as decrease in many bacterial species and diversity (Shannon 2·7, P < 0·01, rRNA) and increase in Sphingomonas spp. as compared to control samples. Furthermore, extra-disinfected copper and PEX samples formed separate clusters in unweighted non-metric multidimensional scaling plot (rRNA) similarly to MWT-treated biofilms of copper (but not PEX) pipes that instead showed higher species diversity (Shannon 4·8, P < 0·05 interaction). Minor chlorine dose addition increased selection pressure and many species were sensitive to chlorination. Pipe material seemed to affect mycobacteria occurrence, and bacterial communities with MWT in copper but not in PEX pipes. This study using rRNA showed that chlorination affects especially active fraction of bacterial communities. Copper and PEX differed by the occurrence of some bacterial members despite similar community profiles. © 2017 The Society for Applied Microbiology.

A modified protocol for RNA extraction from different peach tissues suitable for gene isolation and real-time PCR analysis.

PubMed

Tong, Zhaoguo; Qu, Shenchun; Zhang, Jiyu; Wang, Fei; Tao, Jianmin; Gao, Zhihong; Zhang, Zhen

2012-03-01

RNA extraction is the first step in the study of gene isolation and expression. However, it is difficult to extract high quantity and quality RNA from tissues containing large quantities of polysaccharides and polyphenols. Peach (Prunus persica), in addition to containing high levels of polysaccharides and polyphenols, is a challenging starting material for RNA isolation using a single method because of different amounts of those substances in diverse tissues. Based on three reported methods, we developed a modified RNA isolation protocol to solve this problem, leading to high quality and quantity of total RNA from peach mesocarp tissues of fruits which were sampled from all developmental stages and different storage periods, as well as from other tissues including flowers, leaves, stems, and roots. With our modified method, 28-650 μg of total RNA was routinely obtained from per gram of fresh material, gave at least a 1.16-fold improvement by compared with those isolated by other seven methods. The RNA extracts were successfully used in downstream applications such as RT-PCR, RACE, and real-time PCR.

A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense

PubMed Central

Yu, Nianjun; Zhang, Wei; Xing, Lihua; Xie, Dongmei; Peng, Daiyin

2018-01-01

Acquiring high quality RNA is the basis of plant molecular biology research, plant genetics and other physiological investigations. At present, a large number of nucleotide isolation methods have been exploited or modified, such as commercial kits, CTAB, SDS methods and so on. Due to the nature of different plants, extraction methods vary. Moreover, efficiency of certain approach cannot be guaranteed due to composition of different plants and extracting high quality RNA from plants rich in polysaccharides and polyphenols are often difficult. The physical and chemical properties of polysaccharides which are similar to nucleic acids and other secondary metabolites will be coprecipitated with RNA irreversibly. Therefore, how to remove polysaccharides and other secondary metabolites during RNA extraction is the primary challenge. Dendrobium huoshanense is an Orchidaceae perennial herb that is rich in polysaccharides and other secondary metabolites. By using D. huoshanense as the subject, we improved the method originated from CHAN and made it suitable for plants containing high amount of polysaccharides and polyphenols. The extracted total RNA was clear and non-dispersive, with good integrity and no obvious contamination with DNA and other impurities. And it was also evaluated by gel electrophoresis, nucleic acid quantitative detector and PCR assessment. Thus, as a simple approach, it is suitable and efficient in RNA isolation for plants rich in polysaccharides and polyphenols. PMID:29715304

Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: A quality by design-based approach.

PubMed

Thanki, Kaushik; Zeng, Xianghui; Justesen, Sarah; Tejlmann, Sarah; Falkenberg, Emily; Van Driessche, Elize; Mørck Nielsen, Hanne; Franzyk, Henrik; Foged, Camilla

2017-11-01

Safety and efficacy of therapeutics based on RNA interference, e.g., small interfering RNA (siRNA), are dependent on the optimal engineering of the delivery technology, which is used for intracellular delivery of siRNA to the cytosol of target cells. We investigated the hypothesis that commonly used and poorly tolerated cationic lipids might be replaced with more efficacious and safe lipidoids as the lipid component of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) for achieving more efficient gene silencing at lower and safer doses. However, formulation design of such a complex formulation is highly challenging due to a strong interplay between several contributing factors. Hence, critical formulation variables, i.e. the lipidoid content and siRNA:lipidoid ratio, were initially identified, followed by a systematic quality-by-design approach to define the optimal operating space (OOS), eventually resulting in the identification of a robust, highly efficacious and safe formulation. A 17-run design of experiment with an I-optimal approach was performed to systematically assess the effect of selected variables on critical quality attributes (CQAs), i.e. physicochemical properties (hydrodynamic size, zeta potential, siRNA encapsulation/loading) and the biological performance (in vitro gene silencing and cell viability). Model fitting of the obtained data to construct predictive models revealed non-linear relationships for all CQAs, which can be readily overlooked in one-factor-at-a-time optimization approaches. The response surface methodology further enabled the identification of an OOS that met the desired quality target product profile. The optimized lipidoid-modified LPNs revealed more than 50-fold higher in vitro gene silencing at well-tolerated doses and approx. a twofold increase in siRNA loading as compared to reference LPNs modified with the commonly used cationic lipid dioleyltrimethylammonium propane (DOTAP). Thus, lipidoid-modified LPNs show highly promising prospects for efficient and safe intracellular delivery of siRNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

PubMed Central

Peña-Llopis, Samuel; Brugarolas, James

2014-01-01

Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stemmer, Kerstin; Ellinger-Ziegelbauer, Heidrun; Lotz, Kerstin

2006-11-15

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treatedmore » for 7 days and 6 months with 10 and 1 mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H and E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230{sub 2}.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.« less

XRN 5’→3’ exoribonucleases: Structure, mechanisms and functions

PubMed Central

Nagarajan, Vinay K.; Jones, Christopher I.; Newbury, Sarah F.; Green, Pamela J.

2013-01-01

The XRN family of 5’→3’ exoribonucleases is critical for ensuring the fidelity of cellular RNA turnover in eukaryotes. Highly conserved across species, the family is typically represented by one cytoplasmic enzyme (XRN1/PACMAN or XRN4) and one or more nuclear enzymes (XRN2/RAT1 and XRN3). Cytoplasmic and/or nuclear XRNs have proven to be essential in all organisms tested, and deficiencies can have severe developmental phenotypes, demonstrating that XRNs are indispensable in fungi, plants and animals. XRNs degrade diverse RNA substrates during general RNA decay and function in specialized processes integral to RNA metabolism, such as nonsense-mediated decay (NMD), gene silencing, rRNA maturation, and transcription termination. Here, we review current knowledge of XRNs, highlighting recent work of high impact and future potential. One example is the breakthrough in our understanding of how XRN1 processively degrades 5’ monophosphorylated RNA, revealed by its crystal structure and mutational analysis. The expanding knowledge of XRN substrates and interacting partners is outlined and the functions of XRNs are interpreted at the organismal level using available mutant phenotypes. Finally, three case studies are discussed in more detail to underscore a few of the most exciting areas of research on XRN function: XRN4 involvement in small RNA-associated processes in plants, the roles of XRN1/PACMAN in Drosophila development, and the function of human XRN2 in nuclear transcriptional quality control. This article is part of a Special Issue entitled: RNA Decay Mechanisms. PMID:23517755

A contaminant-free assessment of Endogenous Retroviral RNA in human plasma

PubMed Central

Karamitros, Timokratis; Paraskevis, Dimitrios; Hatzakis, Angelos; Psichogiou, Mina; Elefsiniotis, Ioannis; Hurst, Tara; Geretti, Anna-Maria; Beloukas, Apostolos; Frater, John; Klenerman, Paul; Katzourakis, Aris; Magiorkinis, Gkikas

2016-01-01

Endogenous retroviruses (ERVs) comprise 6–8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination. PMID:27640347

Template-Based Modeling of Protein-RNA Interactions

PubMed Central

Zheng, Jinfang; Kundrotas, Petras J.; Vakser, Ilya A.

2016-01-01

Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes. PMID:27662342

ASSESSING HABITAT QUALITY OF MOUNT HOPE BAY AND NARRAGANSETT BAY USING GROWTH, RNA:DNA, AND FEDDING HABITS OF CAGED JUVENILE WINTER FLOUNDER

EPA Science Inventory

Somatic growth rates, RNA:DNA, and feeding habits of juvenile Pseudopleuronectes americanus (Winter Flounder) were used to asses small-scale spatio-temporal variations in the habitat quality of Mount Hope Bay and Narragan-sett Bay, RI. Three successive caging experiments (14–16 d...

Aspirin can elicit the recurrence of gastric ulcer induced with acetic acid in rats.

PubMed

Wang, Guo-Zhong; Huang, Guo-Ping; Yin, Guo-Li; Zhou, Gang; Guo, Chun-Juan; Xie, Chun-Gang; Jia, Bing-Bing; Wang, Jin-Fu

2007-01-01

This study was supported by grants of New Ideas Capability for Backbone Teachers in Universities of Heilongjiang and of Scientific Research foundation in Qiqihar Medical College. Ulcer recurrence and poor healing may be critically important to the development of serious gastrointestinal complications in patients with long-term non-steroid anti-inflammatory drugs (NSAIDs). The present study is to investigate the effects of aspirin on ulcer recurrence and healing quality and to explore the mechanism. Gastric ulcers were induced with acetic acid in rats; aspirin was administrated by gavage from day 25 to day 54 after ulcer induction. The gastric juice volume, pH, gastric mucus, gastric mucosal blood flow (GMBF) and prostaglandin E(2) (PGE(2)) were measured. The mRNA transcription of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were analyzed with RT-PCR and protein expression with Western blot. The gastric juice volume was significantly increased in aspirin group compared with those of fasting or saline control groups (P<0.01); while the pH, mucus, GMBF and PGE(2) were significantly decreased in aspirin treated rats compared with those of other two groups (P<0.01). COX-2, evaluated with mRNA and protein expression, was significantly augmented in aspirin group compared with others. The quality of ulcer healing (QOUH) in Aspirin group was poorer than that of fasting or saline control groups. Aspirin enhance the recurrence of gastric ulcer. The inhibition of cycloxygenase, mucus secretion and mucosal blood flow may be involved. Aspirin also impair the quality of ulcer healing.

Genome-wide assessment of differential translations with ribosome profiling data

PubMed Central

Xiao, Zhengtao; Zou, Qin; Liu, Yu; Yang, Xuerui

2016-01-01

The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations. PMID:27041671

Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

PubMed Central

Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

2012-01-01

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: quality by design optimization and characterization.

PubMed

Cun, Dongmei; Jensen, Ditte Krohn; Maltesen, Morten Jonas; Bunker, Matthew; Whiteside, Paul; Scurr, David; Foged, Camilla; Nielsen, Hanne Mørck

2011-01-01

Poly(DL-lactide-co-glycolide acid) (PLGA) is an attractive polymer for delivery of biopharmaceuticals owing to its biocompatibility, biodegradability and outstanding controlled release characteristics. The purpose of this study was to understand and define optimal parameters for preparation of small interfering RNA (siRNA)-loaded PLGA nanoparticles by the double emulsion solvent evaporation method and characterize their properties. The experiments were performed according to a 2(5-1) fractional factorial design based on five independent variables: The volume ratio between the inner water phase and the oil phase, the PLGA concentration, the sonication time, the siRNA load and the amount of acetylated bovine serum albumin (Ac-BSA) in the inner water phase added to stabilize the primary emulsion. The effects on the siRNA encapsulation efficiency and the particle size were investigated. The most important factors for obtaining an encapsulation efficiency as high as 70% were the PLGA concentration and the volume ratio whereas the size was mainly affected by the PLGA concentration. The viscosity of the oil phase was increased at high PLGA concentration, which explains the improved encapsulation by stabilization of the primary emulsion and reduction of siRNA leakage to the outer water phase. Addition of Ac-BSA increased the encapsulation efficiency at low PLGA concentrations. The PLGA matrix protected siRNA against nuclease degradation, provided a burst release of surface-localized siRNA followed by a triphasic sustained release for two months. These results enable careful understanding and definition of optimal process parameters for preparation of PLGA nanoparticles encapsulating high amounts of siRNA with immediate and long-term sustained release properties. Copyright © 2010 Elsevier B.V. All rights reserved.

Enhanced Host-Parasite Resistance Based on Down-Regulation of Phelipanche aegyptiaca Target Genes Is Likely by Mobile Small RNA

PubMed Central

Dubey, Neeraj K.; Eizenberg, Hanan; Leibman, Diana; Wolf, Dalia; Edelstein, Menahem; Abu-Nassar, Jackline; Marzouk, Sally; Gal-On, Amit; Aly, Radi

2017-01-01

RNA silencing refers to diverse mechanisms that control gene expression at transcriptional and post-transcriptional levels which can also be used in parasitic pathogens of plants that Broomrapes (Orobanche/Phelipanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops and cause severe negative effects on the yield and yield quality of those crops. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we suggest an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipanche aegyptiaca genes PaACS, PaM6PR, and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing vector and stable expression in transgenic tomato Solanum lycopersicum (Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACS and PaM6PR transcripts from P. aegyptiaca tubercles grown on transgenic tomato or on TRV-infected Nicotiana benthamiana plants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamiana plants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. PMID:28955363

Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing.

PubMed

Aegerter, Sandrine; Jalabert, Bernard; Bobe, Julien

2005-11-01

The mRNA levels of 39 target genes were monitored in unfertilized eggs of 14 rainbow trout sampled the day of ovulation and again 5, 14, and 21 days later. For all 56 collected egg batches, an egg sample was fertilized to estimate egg quality by monitoring embryonic development. Remaining eggs were used for RNA extraction and subsequent real-time PCR analysis. A significant drop of egg quality was observed when eggs were held in the body cavity for 14 or 21 days post-ovulation (dpo). During the same period, eight transcripts (nucleoplasmin or Npm2, ferritin H, tubulin beta, JNK1, cyclin A1, cyclin A2, cathepsin Z, and IGF2) exhibited a differential abundance at one or several collection time(s). Interestingly, we observed higher levels of cyclins A1 and A2 mRNAs in eggs taken 5 days post-ovulation than in eggs taken, from the same females, at the time of ovulation. In addition, seven transcripts exhibited a differential abundance between low quality and high quality eggs. Low quality eggs were characterized by lower levels of Npm2, tubulin beta, and IGF1 transcripts. In contrast, keratins 8 and 18, cathepsin Z, and prostaglandin synthase 2 were more abundant in low quality eggs than in high quality eggs. In this study, we have demonstrated differences in mRNA levels in the rainbow trout egg that are reflective of developmental competence differences induced by post-ovulatory ageing. The putative role of these transcripts in post-ovulatory ageing-induced egg quality defects is discussed with special attention for corresponding cellular functions.

Isolation of high quality RNA from cereal seeds containing high levels of starch.

PubMed

Wang, Guifeng; Wang, Gang; Zhang, Xiaowei; Wang, Fang; Song, Rentao

2012-01-01

Cereals are an important source of food, feed and fuel with a rapidly increasing global demand. However, cereal seeds contain high levels of starch and polysaccharides, making the isolation of high quality RNA extremely difficult. To develop a novel method for extracting high quality total RNA from various starch- and polysaccharides-rich cereal seeds, such as maize, rice, sorghum and wheat. We developed a modified sodium dodecyl sulphate (SDS)/TRIzol method. The combined use of a Tris buffer (pH 9.0) and SDS before TRIzol extraction effectively resolved the problem of seed homogenate solidification in such a buffer. A high concentration of SDS was used separately, not only to promote cell lysis but also to effectively dissolve seed sample containing high levels of starch. Moreover, acid phenol saturated with 0.1  M citrate buffer (pH 4.3) was used to separate RNA from DNAs, proteins and high levels of starch. This rapid protocol was compared with other RNA isolation methods preferentially used for plants rich in polysaccharides and secondary metabolites. Gel electrophoresis analysis indicated that the extracted total RNA had good integrity without apparent DNA contamination. Furthermore, an A₂₆₀/₂₈₀ ratio of approximately 2.0, an A₂₆₀/₂₃₀ ratio of more than 2.0 and RIN values of more than 8.6 indicated that the isolated RNA was of high purity. The isolated RNA was suitable for subsequent molecular manipulations, such as reverse-transcription polymerase chain reaction (PCR), rapid amplification of cDNA ends (RACE) and real-time PCR. The study has described an easy, efficient and highly reproducible method for RNA isolation from various cereal seeds. Copyright © 2011 John Wiley & Sons, Ltd.

Curcumin Rescues a PINK1 Knock Down SH-SY5Y Cellular Model of Parkinson's Disease from Mitochondrial Dysfunction and Cell Death.

PubMed

van der Merwe, Celia; van Dyk, Hayley Christy; Engelbrecht, Lize; van der Westhuizen, Francois Hendrikus; Kinnear, Craig; Loos, Ben; Bardien, Soraya

2017-05-01

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of dopaminergic neurons in the substantia nigra. Mutations in the PINK1 gene result in an autosomal recessive form of early-onset PD. PINK1 plays a vital role in mitochondrial quality control via the removal of dysfunctional mitochondria. The aim of the present study was to create a cellular model of PD using siRNA-mediated knock down of PINK1 in SH-SY5Y neuroblastoma cells The possible protective effects of curcumin, known for its many beneficial properties including antioxidant and anti-inflammatory effects, was tested on this model in the presence and absence of paraquat, an additional stressor. PINK1 siRNA and control cells were separated into four treatment groups: (i) untreated, (ii) treated with paraquat, (iii) pre-treated with curcumin then treated with paraquat, or (iv) treated with curcumin. Various parameters of cellular and mitochondrial function were then measured. The PINK1 siRNA cells exhibited significantly decreased cell viability, mitochondrial membrane potential (MMP), mitochondrial respiration and ATP production, and increased apoptosis. Paraquat-treated cells exhibited decreased cell viability, increased apoptosis, a more fragmented mitochondrial network and decreased MMP. Curcumin pre-treatment followed by paraquat exposure rescued cell viability and increased MMP and mitochondrial respiration in control cells, and significantly decreased apoptosis and increased MMP and maximal respiration in PINK1 siRNA cells. These results highlight a protective effect of curcumin against mitochondrial dysfunction and apoptosis in PINK1-deficient and paraquat-exposed cells. More studies are warranted to further elucidate the potential neuroprotective properties of curcumin.

NetMiner-an ensemble pipeline for building genome-wide and high-quality gene co-expression network using massive-scale RNA-seq samples.

PubMed

Yu, Hua; Jiao, Bingke; Lu, Lu; Wang, Pengfei; Chen, Shuangcheng; Liang, Chengzhi; Liu, Wei

2018-01-01

Accurately reconstructing gene co-expression network is of great importance for uncovering the genetic architecture underlying complex and various phenotypes. The recent availability of high-throughput RNA-seq sequencing has made genome-wide detecting and quantifying of the novel, rare and low-abundance transcripts practical. However, its potential merits in reconstructing gene co-expression network have still not been well explored. Using massive-scale RNA-seq samples, we have designed an ensemble pipeline, called NetMiner, for building genome-scale and high-quality Gene Co-expression Network (GCN) by integrating three frequently used inference algorithms. We constructed a RNA-seq-based GCN in one species of monocot rice. The quality of network obtained by our method was verified and evaluated by the curated gene functional association data sets, which obviously outperformed each single method. In addition, the powerful capability of network for associating genes with functions and agronomic traits was shown by enrichment analysis and case studies. In particular, we demonstrated the potential value of our proposed method to predict the biological roles of unknown protein-coding genes, long non-coding RNA (lncRNA) genes and circular RNA (circRNA) genes. Our results provided a valuable and highly reliable data source to select key candidate genes for subsequent experimental validation. To facilitate identification of novel genes regulating important biological processes and phenotypes in other plants or animals, we have published the source code of NetMiner, making it freely available at https://github.com/czllab/NetMiner.

Extraction and labeling methods for microarrays using small amounts of plant tissue.

PubMed

Stimpson, Alexander J; Pereira, Rhea S; Kiss, John Z; Correll, Melanie J

2009-03-01

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).

Protein-driven RNA nanostructured devices that function in vitro and control mammalian cell fate.

PubMed

Shibata, Tomonori; Fujita, Yoshihiko; Ohno, Hirohisa; Suzuki, Yuki; Hayashi, Karin; Komatsu, Kaoru R; Kawasaki, Shunsuke; Hidaka, Kumi; Yonehara, Shin; Sugiyama, Hiroshi; Endo, Masayuki; Saito, Hirohide

2017-09-14

Nucleic acid nanotechnology has great potential for future therapeutic applications. However, the construction of nanostructured devices that control cell fate by detecting and amplifying protein signals has remained a challenge. Here we design and build protein-driven RNA-nanostructured devices that actuate in vitro by RNA-binding-protein-inducible conformational change and regulate mammalian cell fate by RNA-protein interaction-mediated protein assembly. The conformation and function of the RNA nanostructures are dynamically controlled by RNA-binding protein signals. The protein-responsive RNA nanodevices are constructed inside cells using RNA-only delivery, which may provide a safe tool for building functional RNA-protein nanostructures. Moreover, the designed RNA scaffolds that control the assembly and oligomerization of apoptosis-regulatory proteins on a nanometre scale selectively kill target cells via specific RNA-protein interactions. These findings suggest that synthetic RNA nanodevices could function as molecular robots that detect signals and localize target proteins, induce RNA conformational changes, and programme mammalian cellular behaviour.Nucleic acid nanotechnology has great potential for future therapeutic applications. Here the authors build protein-driven RNA nanostructures that can function within mammalian cells and regulate the cell fate.

Dietary resveratrol supplementation improves meat quality of finishing pigs through changing muscle fiber characteristics and antioxidative status.

PubMed

Zhang, Cheng; Luo, Junqiu; Yu, Bing; Zheng, Ping; Huang, Zhiqing; Mao, Xiangbing; He, Jun; Yu, Jie; Chen, Jiali; Chen, Daiwen

2015-04-01

This study investigated the effects of resveratrol (0, 300, 600 mg/kg) on meat quality, muscle fiber characteristics and antioxidative capacity of finishing pigs. The results showed that resveratrol not only increased m. longissimus dorsi (LM) pH(24 h), a*, crude protein and myoglobin content but also decreased L*(24 h), shear force, drip loss, glycolytic potential, as well as backfat depth, LM lactate dehydrogenase activity and mRNA level. Meanwhile, LM total antioxidative capacity, glutathione peroxidase activity and its mRNA level were increased by resveratrol, while malonaldehyde content was decreased. In addition, resveratrol increased myosin heavy chain (MyHC)IIa mRNA level and decreased MyHCIIb mRNA level, along with decreased myofiber cross-sectional area. In conclusion, these results suggest that resveratrol is an effective feed additive to improve pork quality, and the underlying mechanism may be partly due to the changed muscle fiber characteristics and antioxidative capacity induced by resveratrol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Preparation of RNA from bacteria infected with bacteriophages: a case study from the marine unicellular Synechococcus sp. WH7803 infected by phage S-PM2.

PubMed

Shan, Jinyu; Clokie, Martha

2009-01-01

Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

Effects and Tolerance of Silymarin (Milk Thistle) in Chronic Hepatitis C Virus Infection Patients: A Meta-Analysis of Randomized Controlled Trials

PubMed Central

Zhuang, Liping; Lu, Yunfei; Xu, Qingnian; Chen, Xiaorong

2014-01-01

Objective. This study aimed to evaluate the efficacy and safety of silymarin on chronic hepatitis C virus- (HCV-) infected patients. Methods. Randomized controlled trials (RCTs) of silymarin in chronic HCV-infected patients up to April 1, 2014 were systematically identified in PubMed, Ovid, Web of Science, and Cochrane Library databases. Results. A total of 222 and 167 patients in five RCTs were randomly treated with silymarin (or intravenous silibinin) and placebo, respectively. Serum HCV RNA relatively decreased in patients treated with silymarin compared with those administered with placebo, but no significance was found (P = 0.09). Meta-analysis of patients orally treated with silymarin indicated that the changes of HCV RNA are similar in the two groups (P = 0.19). The effect on alanine aminotransferase (ALT) of oral silymarin is not different from that of placebo (P = 0.45). Improvements in quality-of-life (Short Form-36) in both silymarin and placebo recipients were impressive but relatively identical (P = 0.09). Conclusion. Silymarin is well tolerated in chronic HCV-infected patients. However, no evidence of salutary effects of oral silymarin has yet been reported based on intermediate endpoints (ALT and HCV RNA) in this population. Moreover, intravenous administration of silymarin should be further studied. PMID:25247194

Effects and tolerance of silymarin (milk thistle) in chronic hepatitis C virus infection patients: a meta-analysis of randomized controlled trials.

PubMed

Yang, Zongguo; Zhuang, Liping; Lu, Yunfei; Xu, Qingnian; Chen, Xiaorong

2014-01-01

This study aimed to evaluate the efficacy and safety of silymarin on chronic hepatitis C virus- (HCV-) infected patients. Randomized controlled trials (RCTs) of silymarin in chronic HCV-infected patients up to April 1, 2014 were systematically identified in PubMed, Ovid, Web of Science, and Cochrane Library databases. A total of 222 and 167 patients in five RCTs were randomly treated with silymarin (or intravenous silibinin) and placebo, respectively. Serum HCV RNA relatively decreased in patients treated with silymarin compared with those administered with placebo, but no significance was found (P = 0.09). Meta-analysis of patients orally treated with silymarin indicated that the changes of HCV RNA are similar in the two groups (P = 0.19). The effect on alanine aminotransferase (ALT) of oral silymarin is not different from that of placebo (P = 0.45). Improvements in quality-of-life (Short Form-36) in both silymarin and placebo recipients were impressive but relatively identical (P = 0.09). Silymarin is well tolerated in chronic HCV-infected patients. However, no evidence of salutary effects of oral silymarin has yet been reported based on intermediate endpoints (ALT and HCV RNA) in this population. Moreover, intravenous administration of silymarin should be further studied.

Transcriptomic Profiling and Functional Characterization of Fusion Genes in Recurrent Ovarian Cancer

DTIC Science & Technology

2017-09-01

the enhanced malignancy observed in recurrent disease. In the first year of this proposal we have assembled a cohort of 18 patient matched pairs of...significance and biologic function of prioritized RNA fusion events. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 ...cellularity. 19 cases were identified (Table 1) but one was removed for quality control issues thus leaving a total of 18 cases. Table 1 shows the clinical

The RNA-Seq-based high resolution gene expression atlas of chickpea (Cicer arietinum L.) reveals dynamic spatio-temporal changes associated with growth and development.

PubMed

Kudapa, Himabindu; Garg, Vanika; Chitikineni, Annapurna; Varshney, Rajeev K

2018-04-10

Chickpea is one of the world's largest cultivated food legumes and is an excellent source of high-quality protein to the human diet. Plant growth and development are controlled by programmed expression of a suite of genes at the given time, stage, and tissue. Understanding how the underlying genome sequence translates into specific plant phenotypes at key developmental stages, information on gene expression patterns is crucial. Here, we present a comprehensive Cicer arietinum Gene Expression Atlas (CaGEA) across different plant developmental stages and organs covering the entire life cycle of chickpea. One of the widely used drought tolerant cultivars, ICC 4958 has been used to generate RNA-Seq data from 27 samples at 5 major developmental stages of the plant. A total of 816 million raw reads were generated and of these, 794 million filtered reads after quality control (QC) were subjected to downstream analysis. A total of 15,947 unique number of differentially expressed genes across different pairwise tissue combinations were identified. Significant differences in gene expression patterns contributing in the process of flowering, nodulation, and seed and root development were inferred in this study. Furthermore, differentially expressed candidate genes from "QTL-hotspot" region associated with drought stress response in chickpea were validated. © 2018 The Authors. Plant, Cell & Environment Published by John Wiley & Sons Ltd.

A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines.

PubMed

de Lima, Júlio César; Füller, Thanise Nogueira; de Costa, Fernanda; Rodrigues-Corrêa, Kelly C S; Fett-Neto, Arthur G

2016-01-01

RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid.

PubMed

Mornkham, Tanupat; Wangsomnuk, Preeya Puangsomlee; Fu, Yong-Bi; Wangsomnuk, Pinich; Jogloy, Sanun; Patanothai, Aran

2013-04-29

Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.

The roles of SSU processome components and surveillance factors in the initial processing of human ribosomal RNA

PubMed Central

Sloan, Katherine E.; Bohnsack, Markus T.; Schneider, Claudia; Watkins, Nicholas J.

2014-01-01

During eukaryotic ribosome biogenesis, three of the mature ribosomal (r)RNAs are released from a single precursor transcript (pre-rRNA) by an ordered series of endonucleolytic cleavages and exonucleolytic processing steps. Production of the 18S rRNA requires the removal of the 5′ external transcribed spacer (5′ETS) by endonucleolytic cleavages at sites A0 and A1/site 1. In metazoans, an additional cleavage in the 5′ETS, at site A′, upstream of A0, has also been reported. Here, we have investigated how A′ processing is coordinated with assembly of the early preribosomal complex. We find that only the tUTP (UTP-A) complex is critical for A′ cleavage, while components of the bUTP (UTP-B) and U3 snoRNP are important, but not essential, for efficient processing at this site. All other factors involved in the early stages of 18S rRNA processing that were tested here function downstream from this processing step. Interestingly, we show that the RNA surveillance factors XRN2 and MTR4 are also involved in A′ cleavage in humans. A′ cleavage is largely bypassed when XRN2 is depleted, and we also discover that A′ cleavage is not always the initial processing event in all cell types. Together, our data suggest that A′ cleavage is not a prerequisite for downstream pre-rRNA processing steps and may, in fact, represent a quality control step for initial pre-rRNA transcripts. Furthermore, we show that components of the RNA surveillance machinery, including the exosome and TRAMP complexes, also play key roles in the recycling of excised spacer fragments and degradation of aberrant pre-rRNAs in human cells. PMID:24550520

Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

PubMed

Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John

2016-11-01

Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Enhanced Detection of Bacteria in Environmental Waters: an RNA-based Approach

EPA Science Inventory

Molecular assays (i.e., PCR and qPCR) used in microbial water quality studies often target ribosomal RNA genes (rDNA). However, using DNA as the PCR template does not discriminate between active and dead cells. The use of RNA-based detection methods has recently been proposed as ...

A genome-wide longitudinal transcriptome analysis of the aging model Podospora anserina.

PubMed

Philipp, Oliver; Hamann, Andrea; Servos, Jörg; Werner, Alexandra; Koch, Ina; Osiewacz, Heinz D

2013-01-01

Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations.

SERE: single-parameter quality control and sample comparison for RNA-Seq.

PubMed

Schulze, Stefan K; Kanwar, Rahul; Gölzenleuchter, Meike; Therneau, Terry M; Beutler, Andreas S

2012-10-03

Assessing the reliability of experimental replicates (or global alterations corresponding to different experimental conditions) is a critical step in analyzing RNA-Seq data. Pearson's correlation coefficient r has been widely used in the RNA-Seq field even though its statistical characteristics may be poorly suited to the task. Here we present a single-parameter test procedure for count data, the Simple Error Ratio Estimate (SERE), that can determine whether two RNA-Seq libraries are faithful replicates or globally different. Benchmarking shows that the interpretation of SERE is unambiguous regardless of the total read count or the range of expression differences among bins (exons or genes), a score of 1 indicating faithful replication (i.e., samples are affected only by Poisson variation of individual counts), a score of 0 indicating data duplication, and scores >1 corresponding to true global differences between RNA-Seq libraries. On the contrary the interpretation of Pearson's r is generally ambiguous and highly dependent on sequencing depth and the range of expression levels inherent to the sample (difference between lowest and highest bin count). Cohen's simple Kappa results are also ambiguous and are highly dependent on the choice of bins. For quantifying global sample differences SERE performs similarly to a measure based on the negative binomial distribution yet is simpler to compute. SERE can therefore serve as a straightforward and reliable statistical procedure for the global assessment of pairs or large groups of RNA-Seq datasets by a single statistical parameter.

SERE: Single-parameter quality control and sample comparison for RNA-Seq

PubMed Central

2012-01-01

Background Assessing the reliability of experimental replicates (or global alterations corresponding to different experimental conditions) is a critical step in analyzing RNA-Seq data. Pearson’s correlation coefficient r has been widely used in the RNA-Seq field even though its statistical characteristics may be poorly suited to the task. Results Here we present a single-parameter test procedure for count data, the Simple Error Ratio Estimate (SERE), that can determine whether two RNA-Seq libraries are faithful replicates or globally different. Benchmarking shows that the interpretation of SERE is unambiguous regardless of the total read count or the range of expression differences among bins (exons or genes), a score of 1 indicating faithful replication (i.e., samples are affected only by Poisson variation of individual counts), a score of 0 indicating data duplication, and scores >1 corresponding to true global differences between RNA-Seq libraries. On the contrary the interpretation of Pearson’s r is generally ambiguous and highly dependent on sequencing depth and the range of expression levels inherent to the sample (difference between lowest and highest bin count). Cohen’s simple Kappa results are also ambiguous and are highly dependent on the choice of bins. For quantifying global sample differences SERE performs similarly to a measure based on the negative binomial distribution yet is simpler to compute. Conclusions SERE can therefore serve as a straightforward and reliable statistical procedure for the global assessment of pairs or large groups of RNA-Seq datasets by a single statistical parameter. PMID:23033915

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Complete mitochondrial genome of Skylark, Alauda arvensis (Aves: Passeriformes): the first representative of the family Alaudidae with two extensive heteroplasmic control regions.

PubMed

Qian, Chaoju; Wang, Yuanxiu; Guo, Zhichun; Yang, Jianke; Kan, Xianzhao

2013-06-01

The circular mitochondrial genome of Alauda arvensis is 17,018 bp in length, containing 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and 2 extensive heteroplasmic control regions. All of the genes encoded on the H-strand, with the exceptions of one PCG (nad6) and eight tRNA genes (tRNA(Gln), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), tRNA(Ser(UCN)), tRNA(Pro), and tRNA(Glu)), as found in other birds' mitochondrial genomes. All of these PCGs are initiated with ATG, while stopped by six types of stop codons. All tRNA genes have the potential to fold into typical clover-leaf structure. Two extensive heteroplasmic control regions were found, and more interestingly, a minisatellite of 37 nucleotides (5'-TCAATCCCATTGATTTCATTATATTAGTATAAAGAAA-3') with 6 tandem repeats was detected at the end of CR2.

Comparative biomarker expression and RNA integrity in biospecimens derived from radical retropubic and robot-assisted laparoscopic prostatectomies.

PubMed

Ricciardelli, Carmela; Bianco-Miotto, Tina; Jindal, Shalini; Dodd, Thomas J; Cohen, Penelope A; Marshall, Villis R; Sutherland, Peter D; Samaratunga, Hemamali; Kench, James G; Dong, Ying; Wang, Hong; Clements, Judith A; Risbridger, Gail P; Sutherland, Robert L; Tilley, Wayne D; Horsfall, David J

2010-07-01

Knowledge of preanalytic conditions that biospecimens are subjected to is critically important because novel surgical procedures, tissue sampling, handling, and storage might affect biomarker expression or invalidate tissue samples as analytes for some technologies. We investigated differences in RNA quality, gene expression by quantitative real-time PCR, and immunoreactive protein expression of selected prostate cancer biomarkers between tissues from retropubic radical prostatectomy (RRP) and robot-assisted laparoscopic prostatectomy (RALP). Sections of tissue microarray of 23 RALP and 22 RRP samples were stained with antibodies to androgen receptor (AR) and prostate-specific antigen (PSA) as intersite controls, and 14 other candidate biomarkers of research interest to three laboratories within the Australian Prostate Cancer BioResource tissue banking network. Quantitative real-time PCR was done for AR, PSA (KLK3), KLK2, KLK4, and HIF1A on RNA extracted from five RALP and five RRP frozen tissue cores. No histologic differences were observed between RALP and RRP tissue. Biomarker staining grouped these samples into those with increased (PSA, CK8/18, CKHMW, KLK4), decreased (KLK2, KLK14), or no change in expression (AR, ghrelin, Ki67, PCNA, VEGF-C, PAR2, YB1, p63, versican, and chondroitin 0-sulfate) in RALP compared with RRP tissue. No difference in RNA quality or gene expression was detected between RALP and RRP tissue. Changes in biomarker expression between RALP and RRP tissue exist at the immunoreactive protein level, but the etiology is unclear. Future studies should account for changes in biomarker expression when using RALP tissues, and mixed cohorts of RALP and RRP tissue should be avoided.

miRNA-29a targets COL3A1 to regulate the level of type III collagen in pig.

PubMed

Chuan-Hao, Li; Wei, Chen; Jia-Qing, Hu; Yan-Dong, Wang; Shou-Dong, Wang; Yong-Qing, Zeng; Hui, Wang

2016-10-30

COL3A1 encodes the protein, collagen type III alpha 1, which is an important component of collagen. Collagen can have a considerable effect on the processing quality of meat, and is nutritious. Bioinformatic analysis using Targetscan showed that COL3A1 could be a target gene of miRNA-29a. Moreover, we found that Laiwu pigs have higher levels of type III collagen and lower levels of miRNA-29a than Landrace pigs. Therefore, we hypothesized that miRNA-29a suppresses the expression of COL3A1 by targeting its 3'-UTR. miRNA-29a appears to play an inhibitory role in the regulation of COL3A1 in PK15 cells because of the following: (1) overexpression of miRNA-29a resulted in a significant down-regulation of COL3A1 protein levels (2) overexpression of miRNA-29a significantly decreased the level of COL3A1 mRNA. (3) The activity of a COL3A1 luciferase reporter was significant reduced by miRNA-29a. Furthermore, the levels of miRNA-29a and collagen type III in four tissues in Laiwu and Landrace pigs were consistent with the above observations. In this study, we identified COL3A1 as a direct target for miRNA-29a, which will inform further studies of meat quality. Copyright © 2016 Elsevier B.V. All rights reserved.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

PubMed

Redmond, Latasha C; Pang, Christopher J; Dumur, Catherine; Haar, Jack L; Lloyd, Joyce A

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

PubMed Central

Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

Total rRNA-Seq Analysis Gives Insight into Bacterial, Fungal, Protozoal and Archaeal Communities in the Rumen Using an Optimized RNA Isolation Method

PubMed Central

Elekwachi, Chijioke O.; Wang, Zuo; Wu, Xiaofeng; Rabee, Alaa; Forster, Robert J.

2017-01-01

Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa. PMID:28983291

Total rRNA-Seq Analysis Gives Insight into Bacterial, Fungal, Protozoal and Archaeal Communities in the Rumen Using an Optimized RNA Isolation Method.

PubMed

Elekwachi, Chijioke O; Wang, Zuo; Wu, Xiaofeng; Rabee, Alaa; Forster, Robert J

2017-01-01

Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa.

Production of high quality brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) RNA from isolated populations of rat spinal cord motor neurons obtained by Laser Capture Microdissection (LCM).

PubMed

Mehta, Prachi; Premkumar, Brian; Morris, Renée

2016-08-03

The mammalian central nervous system (CNS) is composed of multiple cellular elements, making it challenging to segregate one particular cell type to study their gene expression profile. For instance, as motor neurons represent only 5-10% of the total cell population of the spinal cord, meaningful transcriptional analysis on these neurons is almost impossible to achieve from homogenized spinal cord tissue. A major challenge faced by scientists is to obtain good quality RNA from small amounts of starting material. In this paper, we used Laser Capture Microdissection (LCM) techniques to identify and isolate spinal cord motor neurons. The present analysis revealed that perfusion with paraformaldehyde (PFA) does not alter RNA quality. RNA integrity numbers (RINs) of tissue samples from rubrospinal tract (RST)-transected, intact spinal cord or from whole spinal cord homogenate were all above 8, which indicates intact, high-quality RNA. Levels of mRNA for brain-derived neurotrophic factor (BDNF) or for its tropomyosin receptor kinase B (TrkB) were not affected by rubrospinal tract (RST) transection, a surgical procedure that deprive motor neurons from one of their main supraspinal input. The isolation of pure populations of neurons with LCM techniques allows for robust transcriptional characterization that cannot be achieved with spinal cord homogenates. Such preparations of pure population of motor neurons will provide valuable tools to advance our understanding of the molecular mechanisms underlying spinal cord injury and neuromuscular diseases. In the near future, LCM techniques might be instrumental to the success of gene therapy for these debilitating conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Assessing the heterogeneity level in lipid nanoparticles for siRNA delivery: size-based separation, compositional heterogeneity, and impact on bioperformance.

PubMed

Zhang, Jingtao; Pei, Yi; Zhang, Hangchun; Wang, Lei; Arrington, Leticia; Zhang, Ye; Glass, Angela; Leone, Anthony M

2013-01-07

A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and in vitro performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60-190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60-150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated, and fractions were studied in-depth for potential presence of polydispersity in size, composition, and in vitro silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semipreparative SEC fractionation that showed similar size, chemical composition, in vitro silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less of a challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the in vitro silencing activity. The impact of LNP size and formulation composition on in vitro performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality.

Ensemble-based prediction of RNA secondary structures.

PubMed

Aghaeepour, Nima; Hoos, Holger H

2013-04-24

Accurate structure prediction methods play an important role for the understanding of RNA function. Energy-based, pseudoknot-free secondary structure prediction is one of the most widely used and versatile approaches, and improved methods for this task have received much attention over the past five years. Despite the impressive progress that as been achieved in this area, existing evaluations of the prediction accuracy achieved by various algorithms do not provide a comprehensive, statistically sound assessment. Furthermore, while there is increasing evidence that no prediction algorithm consistently outperforms all others, no work has been done to exploit the complementary strengths of multiple approaches. In this work, we present two contributions to the area of RNA secondary structure prediction. Firstly, we use state-of-the-art, resampling-based statistical methods together with a previously published and increasingly widely used dataset of high-quality RNA structures to conduct a comprehensive evaluation of existing RNA secondary structure prediction procedures. The results from this evaluation clarify the performance relationship between ten well-known existing energy-based pseudoknot-free RNA secondary structure prediction methods and clearly demonstrate the progress that has been achieved in recent years. Secondly, we introduce AveRNA, a generic and powerful method for combining a set of existing secondary structure prediction procedures into an ensemble-based method that achieves significantly higher prediction accuracies than obtained from any of its component procedures. Our new, ensemble-based method, AveRNA, improves the state of the art for energy-based, pseudoknot-free RNA secondary structure prediction by exploiting the complementary strengths of multiple existing prediction procedures, as demonstrated using a state-of-the-art statistical resampling approach. In addition, AveRNA allows an intuitive and effective control of the trade-off between false negative and false positive base pair predictions. Finally, AveRNA can make use of arbitrary sets of secondary structure prediction procedures and can therefore be used to leverage improvements in prediction accuracy offered by algorithms and energy models developed in the future. Our data, MATLAB software and a web-based version of AveRNA are publicly available at http://www.cs.ubc.ca/labs/beta/Software/AveRNA.

Successful Application of Microarray Technology to Microdissected Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

Coudry, Renata A.; Meireles, Sibele I.; Stoyanova, Radka; Cooper, Harry S.; Carpino, Alan; Wang, Xianqun; Engstrom, Paul F.; Clapper, Margie L.

2007-01-01

The establishment of a reliable method for using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue would provide an opportunity to obtain novel gene expression data from the vast amounts of archived tissue. A custom-designed 22,000 oligonucleotide array was used in the present study to compare the gene expression profile of colonic epithelial cells isolated by laser capture microdissection from FFPE-archived samples with that of the same cell population from matched frozen samples, the preferred source of RNA. Total RNA was extracted from FFPE tissues, amplified, and labeled using the Paradise Reagent System. The quality of the input RNA was assessed by the Bioanalyzer profile, reverse transcriptase-polymerase chain reaction, and agarose gel electrophoresis. The results demonstrate that it is possible to obtain reliable microarray data from FFPE samples using RNA acquired by laser capture microdissection. The concordance between matched FFPE and frozen samples was evaluated and expressed as a Pearson’s correlation coefficient, with values ranging from 0.80 to 0.97. The presence of ribosomal RNA peaks in FFPE-derived RNA was reflected by a high correlation with paired frozen samples. A set of practical recommendations for evaluating the RNA integrity and quality in FFPE samples is reported. PMID:17251338

Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms.

PubMed

Cury, Jaime Aparecido; Seils, Jennifer; Koo, Hyun

2008-01-01

The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W), suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0) and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of RNA per 100 mg of total cell (or biofilm) dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg) of purified RNA per 100 mg of total cell (or biofilm) dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.

Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB

PubMed Central

2012-01-01

Background Ribonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity. Results In this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed. Conclusions This study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen. PMID:23167513

A Search for Gene Fusions/Translocations in Breast Cancer

DTIC Science & Technology

2009-10-01

including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services , Directorate for Information Operations and...RNA isolated from al l experimental samples was assessed for quality and integrity through Bioanalyzer (Agilent) (RNA Integrity Number ≥8) and 2 to...product was assessed for quality and concentration using the Bioanalyzer and libraries w ith a clean, single pe ak (representing a pproximately 300b p

Inhibition and Avoidance of mRNA Degradation by RNA Viruses

PubMed Central

Moon, Stephanie L.; Barnhart, Michael D.; Wilusz, Jeffrey

2012-01-01

The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. Thus, to maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described below. PMID:22626865

R2R--software to speed the depiction of aesthetic consensus RNA secondary structures.

PubMed

Weinberg, Zasha; Breaker, Ronald R

2011-01-04

With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams. We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes. R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file.

siRNA blocking the RAS signalling pathway and inhibits the growth of oesophageal squamous cell carcinoma in nude mice.

PubMed

Wang, Xinjie; Zheng, Yuling; Fan, Qingxia; Zhang, Xudong; Shi, Yonggang

2014-12-01

The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs. Copyright © 2014 John Wiley & Sons, Ltd.

Assessment of the RNASound RNA Sampling Card for the Preservation of Influenza Virus RNA

PubMed Central

Lau, Hilda; Hurt, Aeron C.

2016-01-01

Shipping influenza virus specimens, isolates or purified RNA is normally conducted at ultra-low temperatures using dry ice to ensure minimal degradation of the samples but this is expensive and requires special packaging and shipping conditions. Therefore, alternative methods for shipping influenza viruses or RNA at ambient temperatures would be desirable. The RNASound RNA Sampling Card (FortiusBio LLC, San Diego, CA, USA) is a device that enables specimens or isolates to be applied to a card, whereby viruses are inactivated, while RNA is preserved and purified RNA can also easily be eluted. To evaluate this card, we applied influenza virus cell culture isolate supernatants to either the RNASound card or Whatman Grade No. 1 filter paper (GE Healthcare, Rydalmere, NSW, Australia) and compared the preservation to that of material stored in liquid form. Preservation was tested using influenza A and B viruses at two different storage temperatures [cool (2–8°C) or room temperature (18–22°C)] and these were compared with control material stored at -80°C, for 7, 14, or 28 days. The quality of the RNA recovered was assessed using real time RT-PCR and Sanger sequencing. The RNASound card was effective in preserving influenza RNA at room temperature for up to 28 days, with only a minor change in real-time RT-PCR cycle threshold values for selected gene targets when comparing between viruses applied to the card or stored at -80°C. Similar results were obtained with filter paper, whilst virus in liquid form performed the worst. Nevertheless, as the RNASound card also has the capability to inactivate viruses in addition to preserving RNA at room temperature for many weeks, this makes it feasible to send samples to laboratories using regular mail, and thus avoid the need for expensive shipping conditions requiring biohazard containers and dry ice. Moreover, the quick and simple RNA recovery from the RNASound card allows recipient labs to obtain RNA without the need for special reagents or equipment. PMID:27853455

Up-regulation of VEZT by small activating RNA inhibits the proliferation, invasion and migration of gastric cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Detian; Shang, Liang; Peng, Lipan

Objective: To identify an effective saRNA sequence that can specifically up-regulate VEZT expression and to determine the influence of saRNA had on gastric cancer cell growth, proliferation, invasion and migration. Methods: Three various saRNAs, that target the VEZT gene promoter at different locations relative to the transcription start site were synthesized. A dsControl saRNA was synthesized as a negative control, and a specific shRNA was synthesized to knockdown VEZT and eliminate any off-target effects of the saRNA. Both SGC-7901 and M-28 cells were either transfected with the different saRNAs, or treated with Lipofectamine2000 alone. To determine the most effective saRNA, real-timemore » PCR and Western blot were used to determine the VEZT mRNA and protein content, respectively, of each treatment group. After selection, both cell lines were treated with the chosen saRNA, dsControl or Lipofectamine2000. The saRNA treated cells were divided into two groups: the first group was used immediately in the experiments, and the second group was transfected with shRNA by using RNAi-Mate. The proliferation of cells transfected with saRNA, or saRNA and shRNA, as well as the other control cells, was detected by CCK-8. The invasive and migratory abilities were determined using the transwell chamber assay. Results: We identified the most effective saRNA via real-time PCR and Western blot. The selected saRNA inhibited the growth, invasion and migration of GC cells by specially reactivating VEZT. The real-time PCR and Western blot results showed that treatment with saRNA caused a significant up-regulation of VEZT, and an obvious decrease in the proliferative, invasive and migratory abilities; compared with the control groups (P < 0.01); furthermore, there were no significant differences among the control groups (P > 0.05). This phenomenon provides a theoretical basis for saRNA design and gene therapy for gastric cancer. - Highlights: • saRNA is a brand-new type of small non-coding RNA. It provides a brand-new therapeutic tool for gastric cancer. • In this manuscript, we designed the new saRNA that was effective for VEZT for the first time. • VEZT is a new TSG, and the reactivation through saRNA is never reported before. • It's the first time to report the RNAa in gastric cancer.« less

RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation.

PubMed

Chatterjee, Anirban; Mirer, Paul L; Zaldivar Santamaria, Elvira; Klapperich, Catherine; Sharon, Andre; Sauer-Budge, Alexis F

2010-06-01

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.

Synthetic biology approach for plant protection using dsRNA.

PubMed

Niehl, Annette; Soininen, Marjukka; Poranen, Minna M; Heinlein, Manfred

2018-02-26

Pathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double-stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability and efficient large-scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo dsRNA production systems that rely on DNA transcription and postsynthetic alignment of single-stranded RNA molecules, the phi6 system is based on the replication of dsRNA by an RNA-dependent RNA polymerase, thus allowing production of high-quality, long dsRNA molecules. The phi6 replication complex was reprogrammed to multiply dsRNA sequences homologous to tobacco mosaic virus (TMV) by replacing the coding regions within two of the three phi6 genome segments with TMV sequences and introduction of these constructs into P. syringae together with the third phi6 segment, which encodes the components of the phi6 replication complex. The stable production of TMV dsRNA was achieved by combining all the three phi6 genome segments and by maintaining the natural dsRNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV-derived dsRNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established dsRNA production system enables the broad application of dsRNA molecules as an efficient, highly flexible, nontransgenic and environmentally friendly approach for protecting crops against viruses and other pathogens. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening.

PubMed

Mensah, Mavis; Borzi, Cristina; Verri, Carla; Suatoni, Paola; Conte, Davide; Pastorino, Ugo; Orazio, Fortunato; Sozzi, Gabriella; Boeri, Mattia

2017-10-26

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

Psychrophile spoilers dominate the bacterial microbiome in musculature samples of slaughter pigs.

PubMed

Mann, Evelyne; Wetzels, Stefanie U; Pinior, Beate; Metzler-Zebeli, Barbara U; Wagner, Martin; Schmitz-Esser, Stephan

2016-07-01

The aim of this study was to disentangle the microbial diversity on porcine musculature. The hypervariable V1-V2 region of the 16S rRNA gene was amplified from DNA samples of clinically healthy slaughter pigs (n=8). Pyrosequencing yielded 37,000 quality-controlled reads and a diverse microbiome with 54-159 OTUs per sample was detected. Interestingly, 6 out of 8 samples were strongly dominated by 1-2 highly abundant OTUs (best hits of highly abundant OTUs: Serratia proteamaculans, Pseudomonas syringae, Aeromonas allosaccharophila, Brochothrix thermosphacta, Acidiphilium cryptum and Escherichia coli). In 1g musculature scraping, 3.20E+06 16S rRNA gene copies and 4.45E+01 Enterobacteriaceae rRNA gene copies were detected with qPCR. We conclude that i.) next-generation sequencing technologies help encompass the full content of complex, bacterial contamination, ii.) psychrophile spoilers dominated the microbiota and iii.) E. coli is an effective marker species for pork contamination, as it was one of very few abundant species being present in all samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Grape RNA-Seq analysis pipeline environment

PubMed Central

Knowles, David G.; Röder, Maik; Merkel, Angelika; Guigó, Roderic

2013-01-01

Motivation: The avalanche of data arriving since the development of NGS technologies have prompted the need for developing fast, accurate and easily automated bioinformatic tools capable of dealing with massive datasets. Among the most productive applications of NGS technologies is the sequencing of cellular RNA, known as RNA-Seq. Although RNA-Seq provides similar or superior dynamic range than microarrays at similar or lower cost, the lack of standard and user-friendly pipelines is a bottleneck preventing RNA-Seq from becoming the standard for transcriptome analysis. Results: In this work we present a pipeline for processing and analyzing RNA-Seq data, that we have named Grape (Grape RNA-Seq Analysis Pipeline Environment). Grape supports raw sequencing reads produced by a variety of technologies, either in FASTA or FASTQ format, or as prealigned reads in SAM/BAM format. A minimal Grape configuration consists of the file location of the raw sequencing reads, the genome of the species and the corresponding gene and transcript annotation. Grape first runs a set of quality control steps, and then aligns the reads to the genome, a step that is omitted for prealigned read formats. Grape next estimates gene and transcript expression levels, calculates exon inclusion levels and identifies novel transcripts. Grape can be run on a single computer or in parallel on a computer cluster. It is distributed with specific mapping and quantification tools, but given its modular design, any tool supporting popular data interchange formats can be integrated. Availability: Grape can be obtained from the Bioinformatics and Genomics website at: http://big.crg.cat/services/grape. Contact: david.gonzalez@crg.eu or roderic.guigo@crg.eu PMID:23329413

In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis.

PubMed

Weiss, Victor U; Bliem, Christina; Gösler, Irene; Fedosyuk, Sofiya; Kratzmeier, Martin; Blaas, Dieter; Allmaier, Günter

2016-06-01

Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

PubMed Central

Kruhøffer, Mogens; Dyrskjøt, Lars; Voss, Thorsten; Lindberg, Raija L.P.; Wyrich, Ralf; Thykjaer, Thomas; Orntoft, Torben F.

2007-01-01

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated microRNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis. PMID:17690207

77 FR 74814 - Regulated Navigation Area; Youngs Bay PacifiCorp Sediment Cap; Youngs Bay, Columbia River...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-18

... proposes the establishment of a Regulated Navigation Area (RNA) at the Youngs Bay PacifiCorp property in Astoria, OR. This RNA is necessary to preserve the integrity of an engineered sediment cap as part of an Oregon Department of Environmental Quality (DEQ) required remedial action. This proposed RNA will do so...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

PubMed Central

Fout, G. Shay; Cashdollar, Jennifer L.; Griffin, Shannon M.; Brinkman, Nichole E.; Varughese, Eunice A.; Parshionikar, Sandhya U.

2016-01-01

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water. PMID:26862985

COMAN: a web server for comprehensive metatranscriptomics analysis.

PubMed

Ni, Yueqiong; Li, Jun; Panagiotou, Gianni

2016-08-11

Microbiota-oriented studies based on metagenomic or metatranscriptomic sequencing have revolutionised our understanding on microbial ecology and the roles of both clinical and environmental microbes. The analysis of massive metatranscriptomic data requires extensive computational resources, a collection of bioinformatics tools and expertise in programming. We developed COMAN (Comprehensive Metatranscriptomics Analysis), a web-based tool dedicated to automatically and comprehensively analysing metatranscriptomic data. COMAN pipeline includes quality control of raw reads, removal of reads derived from non-coding RNA, followed by functional annotation, comparative statistical analysis, pathway enrichment analysis, co-expression network analysis and high-quality visualisation. The essential data generated by COMAN are also provided in tabular format for additional analysis and integration with other software. The web server has an easy-to-use interface and detailed instructions, and is freely available at http://sbb.hku.hk/COMAN/ CONCLUSIONS: COMAN is an integrated web server dedicated to comprehensive functional analysis of metatranscriptomic data, translating massive amount of reads to data tables and high-standard figures. It is expected to facilitate the researchers with less expertise in bioinformatics in answering microbiota-related biological questions and to increase the accessibility and interpretation of microbiota RNA-Seq data.

Structural studies of RNA-protein complexes: A hybrid approach involving hydrodynamics, scattering, and computational methods.

PubMed

Patel, Trushar R; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A; Bujnicki, Janusz M

2017-04-15

The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Assessment of Fine Needle Aspiration Feasibility and Specimen Adequacy for Molecular Diagnostics of Benign Vocal Fold Lesions

PubMed Central

Li, Nicole Y. K.; Dailey, Seth; Thibeault, Susan L.

2014-01-01

Objectives/Hypothesis The use of molecular testing is becoming more significant for the diagnosis and classification of disease. The application of fine-needle aspiration (FNA) biopsy as the means of sampling lesions in union with molecular testing could be a powerful combination in laryngology. The objectives of this study were to investigate 1) if FNA was feasible to sample benign vocal fold lesions; 2) if FNA samples provided sufficient RNA quality for molecular analysis; and 3) if gene expression of FNA samples matched paired surgical excised specimens. Study Design Prospective cross-sectional. Methods Fifteen vocal fold specimens were obtained from adult patients undergoing routine surgical removal for benign vocal fold lesions using FNA and surgical excision. Comparisons were made between FNA and excision biopsies for RNA quality. Correlative analysis was completed for RNA expression of nine genes, including decorin (DCN), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), collagen type VI alpha 3 (COL6A3), superoxide dismutase 1 (SOD1), glutathione S-transferase (GST2), collagen type I alpha 2 (COL1A2), ATP binding cassette (ABC), and procollagen I alpha 1 (COL1A1). Results FNA and excision samples demonstrated similar RNA quality (P > 0.05). Per gene expression, four out of nine genes were moderately correlated between the paired samples (P < 0.05). Conclusions FNA of the vocal fold lamina propria is technically feasible to perform. Further improvement in the FNA technology is desirable to optimize RNA quality for reliable gene expression analysis. PMID:23404571

Ultrasound-Targeted Microbubble Destruction to Deliver siRNA Cancer Therapy

PubMed Central

Carson, Andrew R; McTiernan, Charles F; Lavery, Linda; Grata, Michelle; Leng, Xiaoping; Wang, Jianjun; Chen, Xucai; Villanueva, Flordeliza S

2012-01-01

Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound targeted microbubble destruction (UTMD) could be used to enhance delivery of EGFR-directed small inhibitory RNA (siRNA) to murine squamous cell carcinomas. Custom designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling times, relative to controls receiving EGFR siRNA loaded microbubbles but not ultrasound or control siRNA loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof of concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy. PMID:23010078

The Emerging Field of RNA Nanotechnology

PubMed Central

Guo, Peixuan

2011-01-01

RNA can be designed and manipulated just like DNA while having different rules for base-pairing and displaying functions similar to proteins. The large variety of loops and motifs in RNA allow them to fold into numerous complicated structures. This diversity provides a platform for identifying viable building blocks for particle assemblies, substrate binding and manufacture engineering. RNA thermal stability allows production of multivalent nanostructures with defined stoichiometry. Here we review the unique qualities of RNA nanotechnology and their distinct properties inside the body. We describe techniques for constructing RNA nanoparticles from different building blocks and their applications in nanomedicine. Finally, we discuss challenges in predicting and synthesizing RNA and offer some perspectives on the yield and cost of RNA production. PMID:21102465

Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate.

PubMed Central

Ryals, J; Little, R; Bremer, H

1982-01-01

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments. PMID:6179924

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

NASA Astrophysics Data System (ADS)

Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

2018-01-01

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Post-transcriptional inducible gene regulation by natural antisense RNA.

PubMed

Nishizawa, Mikio; Ikeya, Yukinobu; Okumura, Tadayoshi; Kimura, Tominori

2015-01-01

Accumulating data indicate the existence of natural antisense transcripts (asRNAs), frequently transcribed from eukaryotic genes and do not encode proteins in many cases. However, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. A low-copy-number asRNA may post-transcriptionally regulate gene expression with cis-controlling elements on the mRNA. The asRNA itself may act as regulatory RNA in concert with trans-acting factors, including various RNA-binding proteins that bind to cis-controlling elements, microRNAs, and drugs. A novel mechanism that regulates mRNA stability includes the interaction of asRNA with mRNA by hybridization to loops in secondary structures. Furthermore, recent studies have shown that the functional network of mRNAs, asRNAs, and microRNAs finely tunes the levels of mRNA expression. The post-transcriptional mechanisms via these RNA-RNA interactions may play pivotal roles to regulate inducible gene expression and present the possibility of the involvement of asRNAs in various diseases.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

PubMed

Peeters, M; Huang, C L; Vonk, L A; Lu, Z F; Bank, R A; Helder, M N; Doulabi, B Zandieh

2016-11-01

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. © 2016 Peeters et al.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis

PubMed Central

Peeters, M.; Huang, C. L.; Vonk, L. A.; Lu, Z. F.; Bank, R. A.; Doulabi, B. Zandieh

2016-01-01

Objectives Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. PMID:27881439

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA

PubMed Central

Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

2018-01-01

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells. PMID:29535635

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

PubMed

Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

2018-01-01

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.

Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

PubMed Central

Amexis, Georgios; Oeth, Paul; Abel, Kenneth; Ivshina, Anna; Pelloquin, Francois; Cantor, Charles R.; Braun, Andreas; Chumakov, Konstantin

2001-01-01

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest. PMID:11593021

Methods for processing microarray data.

PubMed

Ares, Manuel

2014-02-01

Quality control must be maintained at every step of a microarray experiment, from RNA isolation through statistical evaluation. Here we provide suggestions for analyzing microarray data. Because the utility of the results depends directly on the design of the experiment, the first critical step is to ensure that the experiment can be properly analyzed and interpreted. What is the biological question? What is the best way to perform the experiment? How many replicates will be required to obtain the desired statistical resolution? Next, the samples must be prepared, pass quality controls for integrity and representation, and be hybridized and scanned. Also, slides with defects, missing data, high background, or weak signal must be rejected. Data from individual slides must be normalized and combined so that the data are as free of systematic bias as possible. The third phase is to apply statistical filters and tests to the data to determine genes (1) expressed above background, (2) whose expression level changes in different samples, and (3) whose RNA-processing patterns or protein associations change. Next, a subset of the data should be validated by an alternative method, such as reverse transcription-polymerase chain reaction (RT-PCR). Provided that this endorses the general conclusions of the array analysis, gene sets whose expression, splicing, polyadenylation, protein binding, etc. change in different samples can be classified with respect to function, sequence motif properties, as well as other categories to extract hypotheses for their biological roles and regulatory logic.

Regulatory BC1 RNA in Cognitive Control

ERIC Educational Resources Information Center

Iacoangeli, Anna; Dosunmu, Aderemi; Eom, Taesun; Stefanov, Dimitre G.; Tiedge, Henri

2017-01-01

Dendritic regulatory BC1 RNA is a non-protein-coding (npc) RNA that operates in the translational control of gene expression. The absence of BC1 RNA in BC1 knockout (KO) animals causes translational dysregulation that entails neuronal phenotypic alterations including prolonged epileptiform discharges, audiogenic seizure activity in vivo, and…

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield.

PubMed

Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Márquez, Ascensión; Martínez-Esteso, María José; Luque, Ignacio; Bru-Martínez, Roque

2014-05-01

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 μg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases.

PubMed

Wang, Pan; Qi, Meng; Barboza, Perry; Leigh, Mary Beth; Ungerfeld, Emilio; Selinger, L Brent; McAllister, Tim A; Forster, Robert J

2011-07-01

The rumen is one of the most powerful fibrolytic fermentation systems known. Gene expression analyses, such as reverse transcription PCR (RT-PCR), microarrays, and metatranscriptomics, are techniques that could significantly expand our understanding of this ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results with these procedures. In this study, we successfully isolated high-quality total RNA from the solid phase of ruminal contents by using an improved RNA extraction method. This method is based on liquid nitrogen grinding of whole ruminal solids without microbial detachment and acid guanidinium - phenol - chloroform extraction combined with column purification. Yields of total RNA were as high as 150 µg per g of fresh ruminal content. The typical large subunit/small subunit rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent Technologies) greater than 8.5. By eliminating the detachment step, the resulting RNA was more representative of the complete ecosystem. Our improved method removed a major barrier limiting analysis of rumen microbial function from a gene expression perspective. The polyA-tailed eukaryotic mRNAs obtained have successfully been applied to next-generation sequencing, and metatranscriptomic analysis of the solid fraction of rumen contents revealed abundant sequences related to rumen fungi.

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

PubMed Central

2011-01-01

The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments. PMID:22136293

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format.

PubMed

Salvo-Chirnside, Eliane; Kane, Steven; Kerr, Lorraine E

2011-12-02

The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.

Increased 5S rRNA oxidation in Alzheimer's disease.

PubMed

Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

2012-01-01

It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

Evaluation of RNA quality in fixed and unembedded mouse embryos by different methods.

PubMed

Mu, Yuan; Zhou, Hong; Li, Wenyan; Hu, Lichao; Zhang, Yiting

2013-10-01

Many miRNAs are highly expressed in spatiotemporal and precise tissue-specific patterns in development. Thus it is necessary to examine their expression pattern in mouse embryos. However, embryos from one pregnant mouse are more than enough for expression analysis such as RT-qPCR, which results in reluctant disposal of remaining embryos. Due to the limitation of short sampling time, it is vitally important to quickly preserve samples to ensure the RNA quality. Thus, it is necessary to develop appropriate methods to fix samples in advance. In this study, two fixatives [methanol/DMSO (4:1) and paraformaldehyde] were applied for embryo (12.5 dpc) fixation and two preservatives (methanol and 30% sucrose) were used for fixed embryo preservation. After storage for one month, the skin, skeletal muscle and brain tissues were dissected from the fixed and unembedded embryos. Total RNAs were extracted by TRIzol® reagent and measured by a spectrophotometer, then were subjected to amplify Actb, Hprt, Gapdh, Rnu6, Snord68 and miR-206-3p by RT-qPCR. Embryos fixed in methanol/DMSO and preserved in 100% methanol at -20°C were able to yield at least 349 bp amplifiable RNA. Although paraformaldehyde fixation and 30% sucrose preservation method only yielded amplicons less than 156 bp, it showed a remarkable ability in preserving small RNAs. Snord68 was expressed stably across skin, skeletal muscle and brain tissues like Rnu6, making its possibility as an internal control for qPCR data normalization. Using Snord68 and/or Rnu6 as internal control, we found that the miR-206-3p expression level in skin was about one quarter of its highest level in the skeletal muscle. Therefore, the techniques in this study would be useful for us to reasonably utilize and preserve precious samples. © 2013.

RNA Crystallization

NASA Technical Reports Server (NTRS)

Golden, Barbara L.; Kundrot, Craig E.

2003-01-01

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

Evaluation of the External RNA Controls Consortium (ERCC) reference material using a modified Latin square design.

PubMed

Pine, P Scott; Munro, Sarah A; Parsons, Jerod R; McDaniel, Jennifer; Lucas, Anne Bergstrom; Lozach, Jean; Myers, Timothy G; Su, Qin; Jacobs-Helber, Sarah M; Salit, Marc

2016-06-24

Highly multiplexed assays for quantitation of RNA transcripts are being used in many areas of biology and medicine. Using data generated by these transcriptomic assays requires measurement assurance with appropriate controls. Methods to prototype and evaluate multiple RNA controls were developed as part of the External RNA Controls Consortium (ERCC) assessment process. These approaches included a modified Latin square design to provide a broad dynamic range of relative abundance with known differences between four complex pools of ERCC RNA transcripts spiked into a human liver total RNA background. ERCC pools were analyzed on four different microarray platforms: Agilent 1- and 2-color, Illumina bead, and NIAID lab-made spotted microarrays; and two different second-generation sequencing platforms: the Life Technologies 5500xl and the Illumina HiSeq 2500. Individual ERCC controls were assessed for reproducible performance in signal response to concentration among the platforms. Most demonstrated linear behavior if they were not located near one of the extremes of the dynamic range. Performance issues with any individual ERCC transcript could be attributed to detection limitations, platform-specific target probe issues, or potential mixing errors. Collectively, these pools of spike-in RNA controls were evaluated for suitability as surrogates for endogenous transcripts to interrogate the performance of the RNA measurement process of each platform. The controls were useful for establishing the dynamic range of the assay, as well as delineating the useable region of that range where differential expression measurements, expressed as ratios, would be expected to be accurate. The modified Latin square design presented here uses a composite testing scheme for the evaluation of multiple performance characteristics: linear performance of individual controls, signal response within dynamic range pools of controls, and ratio detection between pairs of dynamic range pools. This compact design provides an economical sample format for the evaluation of multiple external RNA controls within a single experiment per platform. These results indicate that well-designed pools of RNA controls, spiked into samples, provide measurement assurance for endogenous gene expression studies.

RNActive® Technology: Generation and Testing of Stable and Immunogenic mRNA Vaccines.

PubMed

Rauch, Susanne; Lutz, Johannes; Kowalczyk, Aleksandra; Schlake, Thomas; Heidenreich, Regina

2017-01-01

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

Control of rRNA transcription in Escherichia coli.

PubMed Central

Condon, C; Squires, C; Squires, C L

1995-01-01

The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response. PMID:8531889

Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli.

PubMed Central

Gaal, T; Gourse, R L

1990-01-01

rRNA synthesis in Escherichia coli is subject to at least two regulation systems, growth rate-dependent control and stringent control. The inverse correlation between rRNA synthesis rates and guanosine 3'-diphosphate 5'-diphosphate (ppGpp) levels under various physiological conditions has led to the supposition that ppGpp is the mediator of both control mechanisms by inhibiting transcription from rrn P1 promoters. Recently, relA- spoT- strains have been constructed in which both ppGpp synthesis pathways most likely have been removed (M. Cashel, personal communication). We have confirmed that such strains produce no detectable ppGpp and therefore offer a direct means for testing the involvement of ppGpp in the regulation of rRNA synthesis in vivo. Stringent control was determined by measurement of rRNA synthesis after amino acid starvation, while growth rate control was determined by measurement of rRNA synthesis under different nutritional conditions. As expected, the relA- spoT- strain is relaxed for stringent control. However, growth rate-dependent regulation is unimpaired. These results indicate that growth rate regulation can occur in the absence of ppGpp and imply that ppGpp is not the mediator, or at least is not the sole mediator, of growth rate-dependent control. Therefore, growth rate-dependent control and stringent control may utilize different mechanisms for regulating stable RNA synthesis. PMID:2196571

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

PubMed

Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

2006-10-01

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis

USDA-ARS?s Scientific Manuscript database

RNA splicing is crucial to the production of mature messenger RNAs (mRNA). The protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. It is also a peripheral component of the exon-exon junction complex, which assures the quality and availabil...

Switch from translation to RNA replication in a positive-stranded RNA virus

PubMed Central

Gamarnik, Andrea V.; Andino, Raul

1998-01-01

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. PMID:9694795

MicroRNA in Skeletal Muscle: Its Crucial Roles in Signal Proteins, Mus cle Fiber Type, and Muscle Protein Synthesis.

PubMed

Zhang, Jing; Liu, Yu Lan

2017-01-01

Pork is one of the most economical sources of animal protein for human consumption. Meat quality is an important economic trait for the swine industry, which is primarily determined by prenatal muscle development and postnatal growth. Identification of the molecular mechanisms underlying skeletal muscle development is a key priority. MicroRNAs (miRNAs) are a class of small noncoding RNAs that have emerged as key regulators of skeletal muscle development. A number of muscle-related miRNAs have been identified by functional gain and loss experiments in mouse model. However, determining miRNA-mRNA interactions involved in pig skeletal muscle still remains a significant challenge. For a comprehensive understanding of miRNA-mediated mechanisms underlying muscle development, miRNAome analyses of pig skeletal muscle have been performed by deep sequencing. Additionally, porcine miRNA single nucleotide polymorphisms have been implicated in muscle fiber types and meat quality. The present review provides an overview of current knowledge on recently identified miRNAs involved in myogenesis, muscle fiber type and muscle protein metabolism. Undoubtedly, further systematic understanding of the functions of miRNAs in pig skeletal muscle development will be helpful to expand the knowledge of basic skeletal muscle biology and be beneficial for the genetic improvement of meat quality traits. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A biogenesis step upstream of Microprocessor controls miR-17~92 expression

PubMed Central

Du, Peng; Wang, Longfei; Sliz, Piotr; Gregory, Richard I.

2015-01-01

SUMMARY The precise control of miR-17~92 microRNA (miRNA) is essential for normal development and overexpression of certain miRNAs from this cluster is oncogenic. Here we find the relative expression of the six miRNAs processed from the primary (pri-miR-17~92) transcript is dynamically regulated during embryonic stem cell (ESC) differentiation. Pri-miR-17~92 is processed to a biogenesis intermediate, termed ‘progenitor-miRNA’ (pro-miRNA). Pro-miRNA is an efficient substrate for Microprocessor and is required to selectively license production of pre-miR-17, -18a, -19a, 20a, and -19b from this cluster. Two complementary cis-regulatory repression domains within pri-miR-17~92 are required for the blockade of miRNA processing through the formation of an autoinhibitory RNA conformation. The endonuclease CPSF3 (CPSF73), and the Spliceosome-associated ISY1 are responsible for pro-miRNA biogenesis and expression of all miRNAs within the cluster except miR-92. Thus, developmentally regulated pro-miRNA processing is key step controlling miRNA expression and explains the posttranscriptional control of miR-17~92 expression in development. PMID:26255770

Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench

PubMed Central

Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

2017-01-01

Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information. A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. PMID:28289155

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

PubMed

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

RNA-seq methods for identifying differentially expressed gene in human pancreatic islet cells treated with pro-inflammatory cytokines.

PubMed

Li, Bo; Bi, Chang Long; Lang, Ning; Li, Yu Ze; Xu, Chao; Zhang, Ying Qi; Zhai, Ai Xia; Cheng, Zhi Feng

2014-01-01

Type 1 diabetes is a chronic autoimmune disease in which pancreatic beta cells are killed by the infiltrating immune cells as well as the cytokines released by these cells. Many studies indicate that inflammatory mediators have an essential role in this disease. In the present study, we profiled the transcriptome in human islets of langerhans under control conditions or following exposure to the pro-inflammatory cytokines based on the RNA sequencing dataset downloaded from SRA database. After filtered the low-quality ones, the RNA readers was aligned to human genome hg19 by TopHat and then assembled by Cufflinks. The expression value of each transcript was calculated and consequently differentially expressed genes were screened out. Finally, a total of 63 differentially expressed genes were identified including 60 up-regulated and three down-regulated genes. GBP5 and CXCL9 stood out as the top two most up-regulated genes in cytokines treated samples with the log2 fold change of 12.208 and 10.901, respectively. Meanwhile, PTF1A and REG3G were identified as the top two most down-regulated genes with the log2 fold change of -3.759 and -3.606, respectively. Of note, we also found 262 lncRNAs (long non-coding RNA), 177 of which were inferred as novel lncRNAs. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on the specific function of these lncRNA.

The impact of age, biogenesis, and genomic clustering on Drosophila microRNA evolution

PubMed Central

Mohammed, Jaaved; Flynt, Alex S.; Siepel, Adam; Lai, Eric C.

2013-01-01

The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class. PMID:23882112

Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation.

PubMed

Dueck, Hannah; Khaladkar, Mugdha; Kim, Tae Kyung; Spaethling, Jennifer M; Francis, Chantal; Suresh, Sangita; Fisher, Stephen A; Seale, Patrick; Beck, Sheryl G; Bartfai, Tamas; Kuhn, Bernhard; Eberwine, James; Kim, Junhyong

2015-06-09

Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets.

PubMed

Choi, Cheolwon; Yoon, Seulgi; Moon, Hyesu; Bae, Yun-Ui; Kim, Chae-Bin; Diskul-Na-Ayudthaya, Penchatr; Ngu, Trinh Van; Munir, Javaria; Han, JaeWook; Park, Se Bin; Moon, Jong-Seok; Song, Sujung; Ryu, Seongho

2018-04-11

Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method "mirRICH." The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.

Abnormal levels of expression of plasma microRNA-33 in patients with psoriasis.

PubMed

García-Rodríguez, S; Arias-Santiago, S; Orgaz-Molina, J; Magro-Checa, C; Valenzuela, I; Navarro, P; Naranjo-Sintes, R; Sancho, J; Zubiaur, M

2014-06-01

Circulating microRNAs (miRNA) are involved in the posttranscriptional regulation of genes associated with lipid metabolism (miRNA-33) and vascular function and angiogenesis (miRNA-126). The objective of this exploratory study was to measure plasma levels of miRNA-33 and miRNA-126 in patients with plaque psoriasis and evaluate their association with clinical parameters. We studied 11 patients with plaque psoriasis. The median Psoriasis Area Severity Index (PASI) was 13 (interquartile range [IQR], 9-14) and body surface area involvement was 12 (IQR, 11-15). Eleven healthy controls matched for age and sex were also included. We analyzed cardiovascular risk factors and subclinical carotid atheromatosis. Plasma miRNAs were evaluated using quantitative real-time polymerase chain reaction. Carotid intima-media thickness was greater in patients (0.57mm; IQR, 0.54-0.61; n=11) than in controls (0.50mm; IQR, 0.48-0.54; data available for 9 controls) (P=.0055, Mann-Whitney). Expression of miRNA-33 in patients (5.34; IQR, 3.12-7.96; n=11) was significantly higher than in controls (2.33; IQR, 1.71-2.84; only detected in 7 of 11 controls) (P=.0049, Wilcoxon signed rank). No differences in miRNA-126 levels were observed between patients and controls. In patients (n=11), we observed a positive correlation between miRNA-33 and insulin levels (r=0.7289, P=.0109) and a negative correlation between miRNA-126 and carotid intima-media thickness (r=-0.6181, P=.0426). In psoriasis patients plasma levels of lipid and glucose metabolism-related miRNA-33 are increased and correlated with insulin. The study of circulating miRNA-33 in psoriasis may provide new insights about the associated systemic inflammatory abnormalities. Copyright © 2013 Elsevier España, S.L. and AEDV. All rights reserved.

MetaDP: a comprehensive web server for disease prediction of 16S rRNA metagenomic datasets.

PubMed

Xu, Xilin; Wu, Aiping; Zhang, Xinlei; Su, Mingming; Jiang, Taijiao; Yuan, Zhe-Ming

2016-01-01

High-throughput sequencing-based metagenomics has garnered considerable interest in recent years. Numerous methods and tools have been developed for the analysis of metagenomic data. However, it is still a daunting task to install a large number of tools and complete a complicated analysis, especially for researchers with minimal bioinformatics backgrounds. To address this problem, we constructed an automated software named MetaDP for 16S rRNA sequencing data analysis, including data quality control, operational taxonomic unit clustering, diversity analysis, and disease risk prediction modeling. Furthermore, a support vector machine-based prediction model for intestinal bowel syndrome (IBS) was built by applying MetaDP to microbial 16S sequencing data from 108 children. The success of the IBS prediction model suggests that the platform may also be applied to other diseases related to gut microbes, such as obesity, metabolic syndrome, or intestinal cancer, among others (http://metadp.cn:7001/).

pcr: an R package for quality assessment, analysis and testing of qPCR data

PubMed Central

Ahmed, Mahmoud

2018-01-01

Background Real-time quantitative PCR (qPCR) is a broadly used technique in the biomedical research. Currently, few different analysis models are used to determine the quality of data and to quantify the mRNA level across the experimental conditions. Methods We developed an R package to implement methods for quality assessment, analysis and testing qPCR data for statistical significance. Double Delta CT and standard curve models were implemented to quantify the relative expression of target genes from CT in standard qPCR control-group experiments. In addition, calculation of amplification efficiency and curves from serial dilution qPCR experiments are used to assess the quality of the data. Finally, two-group testing and linear models were used to test for significance of the difference in expression control groups and conditions of interest. Results Using two datasets from qPCR experiments, we applied different quality assessment, analysis and statistical testing in the pcr package and compared the results to the original published articles. The final relative expression values from the different models, as well as the intermediary outputs, were checked against the expected results in the original papers and were found to be accurate and reliable. Conclusion The pcr package provides an intuitive and unified interface for its main functions to allow biologist to perform all necessary steps of qPCR analysis and produce graphs in a uniform way. PMID:29576953

CodingQuarry: highly accurate hidden Markov model gene prediction in fungal genomes using RNA-seq transcripts.

PubMed

Testa, Alison C; Hane, James K; Ellwood, Simon R; Oliver, Richard P

2015-03-11

The impact of gene annotation quality on functional and comparative genomics makes gene prediction an important process, particularly in non-model species, including many fungi. Sets of homologous protein sequences are rarely complete with respect to the fungal species of interest and are often small or unreliable, especially when closely related species have not been sequenced or annotated in detail. In these cases, protein homology-based evidence fails to correctly annotate many genes, or significantly improve ab initio predictions. Generalised hidden Markov models (GHMM) have proven to be invaluable tools in gene annotation and, recently, RNA-seq has emerged as a cost-effective means to significantly improve the quality of automated gene annotation. As these methods do not require sets of homologous proteins, improving gene prediction from these resources is of benefit to fungal researchers. While many pipelines now incorporate RNA-seq data in training GHMMs, there has been relatively little investigation into additionally combining RNA-seq data at the point of prediction, and room for improvement in this area motivates this study. CodingQuarry is a highly accurate, self-training GHMM fungal gene predictor designed to work with assembled, aligned RNA-seq transcripts. RNA-seq data informs annotations both during gene-model training and in prediction. Our approach capitalises on the high quality of fungal transcript assemblies by incorporating predictions made directly from transcript sequences. Correct predictions are made despite transcript assembly problems, including those caused by overlap between the transcripts of adjacent gene loci. Stringent benchmarking against high-confidence annotation subsets showed CodingQuarry predicted 91.3% of Schizosaccharomyces pombe genes and 90.4% of Saccharomyces cerevisiae genes perfectly. These results are 4-5% better than those of AUGUSTUS, the next best performing RNA-seq driven gene predictor tested. Comparisons against whole genome Sc. pombe and S. cerevisiae annotations further substantiate a 4-5% improvement in the number of correctly predicted genes. We demonstrate the success of a novel method of incorporating RNA-seq data into GHMM fungal gene prediction. This shows that a high quality annotation can be achieved without relying on protein homology or a training set of genes. CodingQuarry is freely available ( https://sourceforge.net/projects/codingquarry/ ), and suitable for incorporation into genome annotation pipelines.

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses.

PubMed

Holm, Christian K; Rahbek, Stine H; Gad, Hans Henrik; Bak, Rasmus O; Jakobsen, Martin R; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K; Sun, Chenglong; Thomsen, Martin K; Laustsen, Anders; Nielsen, Camilla G; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R

2016-02-19

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

[Control of RNA biosynthesis in rat liver. Some features of RNA biosynthesis during prolonged protein synthesis inhibition].

PubMed

Todorov, I N; Shen, R A; Zheliabovskaia, S M; Galkin, A P

1976-10-01

A drastic inhibition of protein biosynthesis in rat liver in vivo by cycloheximide (CHI) (0.3 mg/100 g of body weight) first caused an increase of RNA synthesis (after 1 hour), which was then followed by its decrease. Partial gradual restoration of the protein synthesis level was shown to be accompanied by a repeated increase of RNA synthesis (12 hs) and its normalisation after 24 hs. The first maximum of RNA synthesis increase in the isolated nuclei system was AU-type RNA synthesis (sensitive to alpha-amanitine), the second one was due to GC-type RNA synthesis (resistant to this toxin). Purified chromatine template activity in the system with E. coli RNA polymerase (by 14%) an hour after CHI treatment, but 3 hrs later was decreased and subsequently restored (12 hrs after CHI injection). The changes of RNA biosynthesis induced by prolonged protein synthesis inhibition suggest the existence of continuous RNA synthesis control in nuclei. This control is realized by translation system using the feed back principle.

MCT1 and MCT4 kinetic of mRNA expression in different tissues after aerobic exercise at maximal lactate steady state workload.

PubMed

de Araujo, G G; Gobatto, C A; de Barros Manchado-Gobatto, F; Teixeira, L Fm; Dos Reis, I Gm; Caperuto, L C; Papoti, M; Bordin, S; Cavaglieri, C R; Verlengia, R

2015-01-01

We evaluate the mRNA expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in skeletal muscle (soleus, red and white gastrocnemius), heart and liver tissues in mice submitted to a single bout of swimming exercise at the maximal lactate steady state workload (MLSSw). After 72 h of MLSS test, the animals were submitted to a swimming exercise session for 25 min at individual MLSSw. Tissues and muscle samples were obtained at rest (control, n=5), immediately (n=5), 5 h (n=5) and 10 h (n=5) after exercise for determination of the MCT1 and MCT4 mRNA expression (RT-PCR). The MCT1 mRNA expression in liver increased after 10 h in relation to the control, immediate and 5 h groups, but the MCT4 remained unchanged. The MCT1 mRNA expression in heart increased by 31 % after 10 h when compared to immediate, but no differences were observed in relation to the control group. No significant differences were observed for red gastrocnemius in MCT1 and MCT4 mRNA expression. However, white gastrocnemius increased MCT1 mRNA expression immediately when compared to rest, 5 and 10 h test groups. In soleus muscle, the MCT1 mRNA expression increased immediately, 5 and 10 h after exercise when compared to the control. In relation to MCT4 mRNA expression, the soleus increased immediately and 10 h after acute exercise when compared to the control group. The soleus, liver and heart were the main tissues that showed improved the MCT1 mRNA expression, indicating its important role in controlling MLSS concentration in mice.

Design of a Temperature-Responsive Transcription Terminator.

PubMed

Roßmanith, Johanna; Weskamp, Mareen; Narberhaus, Franz

2018-02-16

RNA structures regulate various steps in gene expression. Transcription in bacteria is typically terminated by stable hairpin structures. Translation initiation can be modulated by metabolite- or temperature-sensitive RNA structures, called riboswitches or RNA thermometers (RNATs), respectively. RNATs control translation initiation by occlusion of the ribosome binding site at low temperatures. Increasing temperatures destabilize the RNA structure and facilitate ribosome access. In this study, we exploited temperature-responsive RNAT structures to design regulatory elements that control transcription termination instead of translation initiation in Escherichia coli. In order to mimic the structure of factor-independent intrinsic terminators, naturally occurring RNAT hairpins were genetically engineered to be followed by a U-stretch. Functional temperature-responsive terminators (thermoterms) prevented mRNA synthesis at low temperatures but resumed transcription after a temperature upshift. The successful design of temperature-controlled terminators highlights the potential of RNA structures as versatile gene expression control elements.

Circulating miRNAs in acute new-onset atrial fibrillation and their target mRNA network.

PubMed

da Silva, Ananília Medeiros Gomes; de Araújo, Jéssica Nayara Góes; de Oliveira, Katiene Macêdo; Novaes, Ana Eloísa Melo; Lopes, Mariana Borges; de Sousa, Júlio César Vieira; Filho, Antônio Amorim de Araújo; Luchessi, André Ducati; de Rezende, Adriana Augusto; Hirata, Mário Hiroyuki; Silbiger, Vivian Nogueira

2018-04-20

MicroRNAs (miRNAs) are involved in the pathogenesis of atrial fibrillation (AF), acting on development and progression. Our pilot study investigated the expression of six miRNAs and their miRNA-mRNA interactions in patients with acute new-onset AF, well-controlled AF, and normal sinus rhythm (controls). Plasma of acute new-onset AF patients (n = 5) was collected in the emergency room when patients presented with irregular and fast-atrial fibrillation rhythm. Samples from well-controlled AF (n = 16) and control (n =  15) patients were collected during medical appointments following an ECG. Expression of miR-21, miR-133a, miR-133b, miR-150, miR-328, and miR-499 was analyzed by real-time PCR. Ingenuity Pathway Analysis and the TargetScan database identified the top 30 mRNA targets of these miRNA, seeking the miRNA-mRNA interactions in cardiovascular process. Increased expression of miR-133b (1.4-fold), miR-328 (2.0-fold), and miR-499 (2.3-fold) was observed in patients with acute new-onset AF, compared with well-controlled AF and control patients. Decreased expression of miR-21 was seen in patients with well-controlled AF compared to those with acute new-onset AF and controls (0.6-fold). The miRNA-mRNA interaction demonstrated that SMAD7 and FASLG genes were the targets of miR-21, miR-133b, and miR-499 and were directly related to AF, being involved in apoptosis and fibrosis. The miRNAs had different expression profiles dependent on the AF condition, with higher expression in the acute new-onset AF than well-controlled AF. Clinically, this may contribute to an effective assessment for patients, leading to early detection of AF and monitoring to reduce the risk of other serious cardiovascular events. © 2018 Wiley Periodicals, Inc.

R2R - software to speed the depiction of aesthetic consensus RNA secondary structures

PubMed Central

2011-01-01

Background With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams. Results We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes. Conclusions R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file. PMID:21205310

RNA-seq Analysis of Early Hepatic Response to Handling and Confinement Stress in Rainbow Trout

PubMed Central

Liu, Sixin; Gao, Guangtu; Palti, Yniv; Cleveland, Beth M.; Weber, Gregory M.; Rexroad, Caird E.

2014-01-01

Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (R2 = 0.88). Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database. Accession Numbers Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881. Customized Perl Scripts All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author. PMID:24558395

Evaluation of the miRNA-146a and miRNA-155 Expression Levels in Patients with Oral Lichen Planus.

PubMed

Ahmadi-Motamayel, Fatemeh; Bayat, Zeynab; Hajilooi, Mehrdad; Shahryar-Hesami, Soroosh; Mahdavinezhad, Ali; Samie, Lida; Solgi, Ghasem

2017-12-01

Oral Lichen Planus (OLP) is a chronic autoimmune disease that could be considered as a potential premalignant status. To evaluate the miRNA-146a and miRNA-155 expression levels in patients with oral Lichen planus lesions compared to healthy subjects with normal oral mucosa. Forty patients with oral lichen planus and 18 healthy age and gender-matched controls were recruited in this case-control study. Oral lichen planus was diagnosed clinically and pathologically. The expression levels of two miRNAs in peripheral blood samples were determined using commercial TaqMan MicroRNA Assays. Relative quantification of gene expression was calculated by the 2-ΔΔct method. The expression levels of miRNA-146a and miRNA-155 in patients with oral Lichen planus were significantly higher than those of healthy controls. Also, a direct but insignificant correlation was found between miRNA-155 and miRNA-146a expression levels among the patient group. Our findings indicate that miRNA-146a and miRNA-155 could be potential biomarkers for the immunopathogenesis of oral lichen planus.

Associations of circulating plasma microRNAs with age, body mass index and sex in a population-based study.

PubMed

Ameling, Sabine; Kacprowski, Tim; Chilukoti, Ravi Kumar; Malsch, Carolin; Liebscher, Volkmar; Suhre, Karsten; Pietzner, Maik; Friedrich, Nele; Homuth, Georg; Hammer, Elke; Völker, Uwe

2015-10-14

Non-cellular blood circulating microRNAs (plasma miRNAs) represent a promising source for the development of prognostic and diagnostic tools owing to their minimally invasive sampling, high stability, and simple quantification by standard techniques such as RT-qPCR. So far, the majority of association studies involving plasma miRNAs were disease-specific case-control analyses. In contrast, in the present study, plasma miRNAs were analysed in a sample of 372 individuals from a population-based cohort study, the Study of Health in Pomerania (SHIP). Quantification of miRNA levels was performed by RT-qPCR using the Exiqon Serum/Plasma Focus microRNA PCR Panel V3.M covering 179 different miRNAs. Of these, 155 were included in our analyses after quality-control. Associations between plasma miRNAs and the phenotypes age, body mass index (BMI), and sex were assessed via a two-step linear regression approach per miRNA. The first step regressed out the technical parameters and the second step determined the remaining associations between the respective plasma miRNA and the phenotypes of interest. After regressing out technical parameters and adjusting for the respective other two phenotypes, 7, 15, and 35 plasma miRNAs were significantly (q < 0.05) associated with age, BMI, and sex, respectively. Additional adjustment for the blood cell parameters identified 12 and 19 miRNAs to be significantly associated with age and BMI, respectively. Most of the BMI-associated miRNAs likely originate from liver. Sex-associated differences in miRNA levels were largely determined by differences in blood cell parameters. Thus, only 7 as compared to originally 35 sex-associated miRNAs displayed sex-specific differences after adjustment for blood cell parameters. These findings emphasize that circulating miRNAs are strongly impacted by age, BMI, and sex. Hence, these parameters should be considered as covariates in association studies based on plasma miRNA levels. The established experimental and computational workflow can now be used in future screening studies to determine associations of plasma miRNAs with defined disease phenotypes.

Significance of biological resource collection and tumor tissue bank creation.

PubMed

Yu, Ying-Yan; Zhu, Zheng-Gang

2010-01-15

Progress in the molecular oncology of gastrointestinal carcinomas depends on high quality cancer tissues for research. Recent acceleration on new technological platforms as well as the "omics" revolution increases the demands on tissues and peripheral blood for research at the DNA, mRNA and protein levels. Tissue bank creation emerges as a priority. Tumor tissue banks are facilities that are organized to collect, store and distribute samples of tumor and normal tissue for further use in basic and translational cancer research. The samples are generally obtained immediately after excision, prior to fixation, to ensure optimal preservation of proteins and nucleic acids. It is possible for surgeons or pathologists to collect fresh tissue prospectively during their routine dissection procedures. Most tissue banks are "project-driven" tumor banks, which are specialized collections of tumor samples on which their research is based. Systematic collection of all available tumor tissue is much rarer. High quality tissue banks need the collaboration of clinicians and basic scientists, but also the informed consent of patients and ethical approval. Through the standard operation procedure, snap frozen fresh tissue collection, storage and quality control for cryopreserved tissues are the pivotal factors on tissue bank construction and maintaining. The purpose of the tissue bank creation is enhancing the quality and speed on both the basic and translational research on gastrointestinal cancer. The quality assurance and quality control are handled based on reviewing HE staining slides or touch imprint cytology by pathologists.

Capture and quality control mechanisms for ATP binding

PubMed Central

Li, Li; Martinis, Susan A.

2013-01-01

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly-casting mechanism that acts up on the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates. PMID:23276298

EOL-1, the Homolog of the Mammalian Dom3Z, Regulates Olfactory Learning in C. elegans

PubMed Central

Shen, Yu; Zhang, Jiangwen; Calarco, John A.

2014-01-01

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs. Mutating the residues of Dom3Z that are critical for its enzymatic activity, and the equivalent residues in EOL-1, abolishes the function of these proteins in learning. Together, our results provide insights into the function of EOL-1/Dom3Z and suggest that its activity in pre-mRNA quality control is involved in neural plasticity. PMID:25274815

Increasing seed size and quality by manipulating BIG SEEDS1 in legume species

PubMed Central

Ge, Liangfa; Yu, Jianbin; Wang, Hongliang; Luth, Diane; Bai, Guihua; Wang, Kan

2016-01-01

Plant organs, such as seeds, are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that have been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a conserved role for the BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods, and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight, and amino acid content. Our results provide a strategy for the increase in yield and seed quality in legumes. PMID:27791139

Capture and quality control mechanisms for adenosine-5'-triphosphate binding.

PubMed

Li, Li; Martinis, Susan A; Luthey-Schulten, Zaida

2013-04-24

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly casting mechanism that acts upon the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates.

Library construction for next-generation sequencing: Overviews and challenges

PubMed Central

Head, Steven R.; Komori, H. Kiyomi; LaMere, Sarah A.; Whisenant, Thomas; Van Nieuwerburgh, Filip; Salomon, Daniel R.; Ordoukhanian, Phillip

2014-01-01

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed. PMID:24502796

Comparison of alternative approaches for analysing multi-level RNA-seq data

PubMed Central

Mohorianu, Irina; Bretman, Amanda; Smith, Damian T.; Fowler, Emily K.; Dalmay, Tamas

2017-01-01

RNA sequencing (RNA-seq) is widely used for RNA quantification in the environmental, biological and medical sciences. It enables the description of genome-wide patterns of expression and the identification of regulatory interactions and networks. The aim of RNA-seq data analyses is to achieve rigorous quantification of genes/transcripts to allow a reliable prediction of differential expression (DE), despite variation in levels of noise and inherent biases in sequencing data. This can be especially challenging for datasets in which gene expression differences are subtle, as in the behavioural transcriptomics test dataset from D. melanogaster that we used here. We investigated the power of existing approaches for quality checking mRNA-seq data and explored additional, quantitative quality checks. To accommodate nested, multi-level experimental designs, we incorporated sample layout into our analyses. We employed a subsampling without replacement-based normalization and an identification of DE that accounted for the hierarchy and amplitude of effect sizes within samples, then evaluated the resulting differential expression call in comparison to existing approaches. In a final step to test for broader applicability, we applied our approaches to a published set of H. sapiens mRNA-seq samples, The dataset-tailored methods improved sample comparability and delivered a robust prediction of subtle gene expression changes. The proposed approaches have the potential to improve key steps in the analysis of RNA-seq data by incorporating the structure and characteristics of biological experiments. PMID:28792517

Heart failure-associated changes in RNA splicing of sarcomere genes.

PubMed

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W K; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L; Bisping, Egbert; Pieske, Burkert; dos Remedios, Cristobal G; Pu, William T

2010-04-01

Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1▿

PubMed Central

Zhan, Sien; Li, Jinming; Xu, Ruihuan; Wang, Lunan; Zhang, Kuo; Zhang, Rui

2009-01-01

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5′ terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4°C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA. PMID:19494069

Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1.

PubMed

Zhan, Sien; Li, Jinming; Xu, Ruihuan; Wang, Lunan; Zhang, Kuo; Zhang, Rui

2009-08-01

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5' terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4 degrees C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA.

Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems.

PubMed

Silva, D V; Branco, S M J; Holanda, I S A; Royaert, S; Motamayor, J C; Marelli, J P; Corrêa, R X

2016-03-04

Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.

Exosomal miR-34s panel as potential novel diagnostic and prognostic biomarker in patients with hepatoblastoma.

PubMed

Jiao, Chenwei; Jiao, Xiaohu; Zhu, Anzhi; Ge, Juntao; Xu, Xiaoqing

2017-04-01

The aim of this study is to identify the diagnostic values of serum exosomal miRNA-34s of patients with HB in a large Asian group and explore the prognostic value of the exosomal miRNA-34s panel compared with other risk factors. We retrospectively reviewed 89 children with HB. Among these patients, 63 patients were included as training group to build the diagnostic model for HB. 26 patients were defined as the validation group. The expressions of miRNA-34s were detected by real-time PCR. The comparison of diagnostic and prognostic performance of serum exosomal miRNA-34s was measured using the area under ROC curve (AUC). For patients in the training group, expression of miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with control group in serum exosomes. Between HB training group and the control group, exosomal miRNA-34a, miRNA-34b and miRNA-34c had no significant differences compared with the AFP level in diagnosing HB. The performance of the exosomal miRNA-34s panel in differentiating the HB training group from the control group was superior to the AFP level. The value of the exosomal miRNA-34s panel in predicting prognosis of patients with HB was superior to other risk factors in both training group and validation group. In this study, we found that the expression of exosomal miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with the control group, and we confirmed the exosomal miRNA-34s panel could be defined as a diagnostic and prognostic biomarker for patients with HB. Level II. Retrospective Study. Copyright © 2017 Elsevier Inc. All rights reserved.

RNA-Seq of Circulating Tumor Cells in Stage II-III Breast Cancer.

PubMed

Lang, Julie E; Ring, Alexander; Porras, Tania; Kaur, Pushpinder; Forte, Victoria A; Mineyev, Neal; Tripathy, Debu; Press, Michael F; Campo, Daniel

2018-06-04

We characterized the whole transcriptome of circulating tumor cells (CTCs) in stage II-III breast cancer to evaluate correlations with primary tumor biology. CTCs were isolated from peripheral blood (PB) via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). CTCs, PB, and fresh tumors were profiled using RNA-seq. Formalin-fixed, paraffin-embedded (FFPE) tumors were subjected to RNA-seq and NanoString PAM50 assays with risk of recurrence (ROR) scores. CTCs were detected in 29/33 (88%) patients. We selected 21 cases to attempt RNA-seq (median number of CTCs = 9). Sixteen CTC samples yielded results that passed quality-control metrics, and these samples had a median of 4,311,255 uniquely mapped reads (less than PB or tumors). Intrinsic subtype predicted by comparing estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) versus PAM50 for FFPE tumors was 85% concordant. However, CTC RNA-seq subtype assessed by the PAM50 classification genes was highly discordant, both with the subtype predicted by ER/PR/HER2 and by PAM50 tumors. Two patients died of metastatic disease, both of whom had high ROR scores and high CTC counts. We identified significant genes, canonical pathways, upstream regulators, and molecular interaction networks comparing CTCs by various clinical factors. We also identified a 75-gene signature with highest expression in CTCs and tumors taken together that was prognostic in The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium datasets. It is feasible to use RNA-seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics.

The problems and promise of DNA barcodes for species diagnosis of primate biomaterials

PubMed Central

Lorenz, Joseph G; Jackson, Whitney E; Beck, Jeanne C; Hanner, Robert

2005-01-01

The Integrated Primate Biomaterials and Information Resource (www.IPBIR.org) provides essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing DNA and RNA derived from primate cell cultures. The IPBIR uses mitochondrial cytochrome c oxidase subunit I sequences to verify the identity of samples for quality control purposes in the accession, cell culture, DNA extraction processes and prior to shipping to end users. As a result, IPBIR is accumulating a database of ‘DNA barcodes’ for many species of primates. However, this quality control process is complicated by taxon specific patterns of ‘universal primer’ failure, as well as the amplification or co-amplification of nuclear pseudogenes of mitochondrial origins. To overcome these difficulties, taxon specific primers have been developed, and reverse transcriptase PCR is utilized to exclude these extraneous sequences from amplification. DNA barcoding of primates has applications to conservation and law enforcement. Depositing barcode sequences in a public database, along with primer sequences, trace files and associated quality scores, makes this species identification technique widely accessible. Reference DNA barcode sequences should be derived from, and linked to, specimens of known provenance in web-accessible collections in order to validate this system of molecular diagnostics. PMID:16214744

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies.

PubMed

Hoyle, Nathaniel P; Castelli, Lydia M; Campbell, Susan G; Holmes, Leah E A; Ashe, Mark P

2007-10-08

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

Kid depletion in mouse oocytes associated with multinucleated blastomere formation and inferior embryo development.

PubMed

Egashira, Akiyoshi; Yamauchi, Nobuhiko; Islam, Md Rashedul; Yamagami, Kazuki; Tanaka, Asami; Suyama, Hikaru; El-Sayed, El-Sharawy Mohamed; Tabata, Shoji; Kuramoto, Takashi

2016-08-01

This study investigated the knockdown (KD) of Kid on maturation developmental competence and multinucleation of mouse germinal vesicle (GV) oocytes after parthenogenetic activation. Data revealed that Kid messenger RNA (mRNA) was expressed in GV and MII stage oocyte and 1- and 2-cell embryos. Additionally, Kid mRNA expression in the Kid KD group decreased by nearly 46% compared to the control small interfering RNA (siRNA) groups. The rate of multinucleated embryos in the Kid KD group (52.4%) was significantly higher (P < 0.05) than the control siRNA group (4.7%). Finally, the developmental rates were significantly lower in the Kid siRNA group at > 4-cell stage (28.6% vs. 53.5%) and the blastocyst stage (2.4% vs. 23.3%) compared to the control siRNA groups. Suppression of Kid using siRNA caused multinucleation in early embryos with high frequency and it may increase 2- to 4-cell arrested embryos and reduce the developmental competence to blastocyst. © 2016 Japanese Society of Animal Science.

RNA interfering molecule delivery from in situ forming biodegradable hydrogels for enhancement of bone formation in rat calvarial bone defects.

PubMed

Nguyen, Minh K; Jeon, Oju; Dang, Phuong N; Huynh, Cong T; Varghai, Davood; Riazi, Hooman; McMillan, Alexandra; Herberg, Samuel; Alsberg, Eben

2018-06-06

RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Assembly of RNA nanostructures on supported lipid bilayers

NASA Astrophysics Data System (ADS)

Dabkowska, Aleksandra P.; Michanek, Agnes; Jaeger, Luc; Rabe, Michael; Chworos, Arkadiusz; Höök, Fredrik; Nylander, Tommy; Sparr, Emma

2014-12-01

The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces. Electronic supplementary information (ESI) available: Table with sequences of tRNA units used in this study; schematic structures of the RNA polyhedron and its building blocks; gel electrophoresis characterization of the RNA polyhedron and squares; AFM characterization of RNA tectosquare; schematic structures of RNA-9 and RNA-10 and their association with lipid bilayers; QCM-D frequency and dissipation data (as function of time) for adsorption of RNA polyhedrons, RNA squares and RNA9-10 TIRF images of RNA with Gelstar after photobleaching with analysis; Correlation plot in change of shear viscosity for TS3 and TO3-4 models for the stoichiometry of TS; QCM-D dissipation data for the sequential experiment in Fig. 5a; QCM-D and for the assembly of building blocks at the bilayer scaffold at varying bulk concentrations; QCM-D of adsorption of TS3. See DOI: 10.1039/c4nr05968a

Radiation Exposure Alters Expression of Metabolic Enzyme Genes In Mice

NASA Technical Reports Server (NTRS)

Wotring, Virginia E.; Mangala, L. S.; Zhang, Y.; Wu, H.

2010-01-01

Most pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Because of the importance of the liver in drug metabolism it is important to understand the effects of spaceflight on the enzymes of the liver. Exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. This study is an effort to examine the effects of adaptive mechanisms that may be triggered by early exposure to low radiation doses. Using procedures approved by the JSC Animal Care & Use Committee, C57 male mice were exposed to Cs-137 in groups: controls, low dose (50 mGy), high dose (6Gy) and a fourth group that received both radiation doses separated by 24 hours. Animals were anesthetized and sacrificed 4 hours after their last radiation exposure. Livers were removed immediately and flash-frozen in liquid nitrogen. Tissue was homogenized, RNA extracted and purified (Absolutely RNA, Agilent). Quality of RNA samples was evaluated (Agilent Bioanalyzer 2100). Complementary DNA was prepared from high-quality RNA samples, and used to run RT-qPCR screening arrays for DNA Repair and Drug Metabolism (SuperArray, SABiosciences/Qiagen; BioRad Cfx96 qPCR System). Of 91 drug metabolism genes examined, expression of 7 was altered by at least one treatment condition. Genes that had elevated expression include those that metabolize promethazine and steroids (4-8-fold), many that reduce oxidation products, and one that reduces heavy metal exposure (greater than 200-fold). Of the 91 DNA repair and general metabolism genes examined, expression of 14 was altered by at least one treatment condition. These gene expression changes are likely homeostatic and could lead to development of new radioprotective countermeasures.

QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

PubMed

Zhao, Shanrong; Xi, Li; Quan, Jie; Xi, Hualin; Zhang, Ying; von Schack, David; Vincent, Michael; Zhang, Baohong

2016-01-08

RNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists. By combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines. The high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.

External RNA Controls Consortium Beta Version Update.

PubMed

Lee, Hangnoh; Pine, P Scott; McDaniel, Jennifer; Salit, Marc; Oliver, Brian

2016-01-01

Spike-in RNAs are valuable controls for a variety of gene expression measurements. The External RNA Controls Consortium developed test sets that were used in a number of published reports. Here we provide an authoritative table that summarizes, updates, and corrects errors in the test version that ultimately resulted in the certified Standard Reference Material 2374. We have noted existence of anti-sense RNA controls in the material, corrected sub-pool memberships, and commented on control RNAs that displayed inconsistent behavior.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis.

PubMed

Spangler, Jacob B; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression.

Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

PubMed Central

Spangler, Jacob B.; Feltus, Frank Alex

2013-01-01

Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near α duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near α duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

Preparation of Total RNA from Fission Yeast.

PubMed

Bähler, Jürg; Wise, Jo Ann

2017-04-03

Treatment with hot phenol breaks open fission yeast cells and begins to strip away bound proteins from RNA. Deproteinization is completed by multiple extractions with chloroform/isoamyl alcohol and separation of the aqueous and organic phases using MaXtract gel, an inert material that acts as a physical barrier between the phases. The final step is concentration of the RNA by ethanol precipitation. The protocol can be used to prepare RNA from several cultures grown in parallel, but it is important not to process too many samples at once because delays can be detrimental to RNA quality. A reasonable number of samples to process at once would be three to four for microarray or RNA sequencing analyses and six for preliminary investigations of mutants implicated in RNA metabolism. © 2017 Cold Spring Harbor Laboratory Press.

Formaldehyde substitute fixatives: effects on nucleic acid preservation.

PubMed

Moelans, Cathy B; Oostenrijk, Daphne; Moons, Michiel J; van Diest, Paul J

2011-11-01

In surgical pathology, formalin-fixed paraffin-embedded tissues are increasingly being used as a source of DNA and RNA for molecular assays in addition to histopathological evaluation. However, the commonly used formalin fixative is carcinogenic, and its crosslinking impairs DNA and RNA quality. The suitability of three new presumably less toxic, crosslinking (F-Solv) and non-crosslinking (FineFIX, RCL2) alcohol-based fixatives was tested for routine molecular pathology in comparison with neutral buffered formalin (NBF) as gold standard. Size ladder PCR, epidermal growth factor receptor sequence analysis, microsatellite instability (MSI), chromogenic (CISH), fluorescence in situ hybridisation (FISH) and qPCR were performed. The alcohol-based non-crosslinking fixatives (FineFIX and RCL2) resulted in a higher DNA yield and quality compared with crosslinking fixatives (NBF and F-Solv). Size ladder PCR resulted in a shorter amplicon size (300 bp) for both crosslinking fixatives compared with the non-crosslinking fixatives (400 bp). All four fixatives were directly applicable for MSI and epidermal growth factor receptor sequence analysis. All fixatives except F-Solv showed clear signals in CISH and FISH. RNA yield and quality were superior after non-crosslinking fixation. qPCR resulted in lower Ct values for RCL2 and FineFIX. The alcohol-based non-crosslinking fixatives performed better than crosslinking fixatives with regard to DNA and RNA yield, quality and applicability in molecular diagnostics. Given the higher yield, less starting material may be necessary, thereby increasing the applicability of biopsies for molecular studies.

MicroRNA Expression Profiles as Biomarkers of Minor Salivary Gland Inflammation and Dysfunction in Sjögren's Syndrome

PubMed Central

Alevizos, Ilias; Alexander, Stefanie; Turner, R. James; Illei, Gabor G.

2013-01-01

Objective MicroRNA reflect physiologic and pathologic processes and may be used as biomarkers of concurrent pathophysiologic events in complex settings such as autoimmune diseases. We generated microRNA microarray profiles from the minor salivary glands of control subjects without Sjögren's syndrome (SS) and patients with SS who had low-grade or high-grade inflammation and impaired or normal saliva production, to identify microRNA patterns specific to salivary gland inflammation or dysfunction. Methods MicroRNA expression profiles were generated by Agilent microRNA arrays. We developed a novel method for data normalization by identifying housekeeping microRNA. MicroRNA profiles were compared by unsupervised mathematical methods to test how well they distinguish between control subjects and various subsets of patients with SS. Several bioinformatics methods were used to predict the messenger RNA targets of the differentially expressed microRNA. Results MicroRNA expression patterns accurately distinguished salivary glands from control subjects and patients with SS who had low-degree or high-degree inflammation. Using real-time quantitative polymerase chain reaction, we validated 2 microRNA as markers of inflammation in an independent cohort. Comparing microRNA from patients with preserved or low salivary flow identified a set of differentially expressed microRNA, most of which were up-regulated in the group with decreased salivary gland function, suggesting that the targets of microRNA may have a protective effect on epithelial cells. The predicted biologic targets of microRNA associated with inflammation or salivary gland dysfunction identified both overlapping and distinct biologic pathways and processes. Conclusion Distinct microRNA expression patterns are associated with salivary gland inflammation and dysfunction in patients with SS, and microRNA represent a novel group of potential biomarkers. PMID:21280008

Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli.

PubMed Central

Dennis, P P

1977-01-01

The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate. PMID:320185

Dependence of RNA:DNA ratios and Fulton’s K condition indices on environmental characteristics of plaice and dab nursery grounds

NASA Astrophysics Data System (ADS)

De Raedemaecker, F.; Brophy, D.; O'Connor, I.; O'Neill, B.

2012-02-01

This field study showed a lack of a correlation between a morphometric (Fulton's K) and biochemical (RNA:DNA ratio) condition index in juvenile plaice ( Pleuronectes platessa) and dab ( Limanda limanda) studied to assess habitat quality in four sandy beach nursery grounds in Galway Bay, Ireland. Based on monthly surveys from June to September in 2008 and 2009, fish growth, indicated by RNA:DNA ratios and Fulton's K, displayed considerable spatio-temporal variability. Site-related patterns in Fulton's K for plaice and dab were consistent between years whereas RNA:DNA ratios displayed annual and interspecific variability among nursery habitats. This indicates a higher sensitivity of RNA:DNA ratios to short-term environmental fluctuations which is not apparent in Fulton's K measurements of juvenile flatfish. Generalized Additive Modelling (GAM) revealed non-linear relationships between the condition indices and (biotic and abiotic) habitat characteristics as well as diet features, derived from gut content analyses. Density of predators, sediment grain size and salinity were the most important predictors of both condition indices. Temperature also affected condition indices in dab whereas plaice condition indices varied with depth. Diet features did not contribute to the explained variability in the models predicting RNA:DNA ratios whereas certain prey groups significantly improved the explained variability in the models predicting Fulton's K of plaice and dab. The value of both indices for assessing fish condition and habitat quality in field studies is discussed. These findings aid understanding of the biological and physical mechanisms promoting fast growth and high survival which will help to identify high quality nursery areas for juvenile plaice and dab.

Effect of a soy protein-based diet on ribonucleic acid metabolism in the small intestinal mucosa of goat kids.

PubMed

Schönhusen, U; Kuhla, S; Zitnan, R; Wutzke, K D; Huber, K; Moors, S; Voigt, J

2007-05-01

This study was designed to investigate the effect of soy protein inclusion in milk replacer diets for goat kids on protein, RNA, and DNA contents in small intestinal mucosa, on the importance of RNA biosynthesis from dietary RNA precursors for mucosal RNA synthesis, and on the activities of enzymes involved in nucleotide degradation in small intestinal mucosa. Diets were based on cow's milk. In the control group, 35% of the milk protein was replaced by casein (CN) protein, and in the soy group (SPAA), the same amount of milk protein was replaced by soy protein supplemented with essential AA known to be at lower concentrations in soy than in CN (Thr, Val, Ile, Leu, His, Lys, Met). Diets were isonitrogenous and isoenergetic. At 47 d of age, goats were harvested and samples of proximal, middle, and distal jejunal mucosa were collected 5 h after feeding 15N-labeled RNA from yeast (13 mg/kg of body weight). Growth and feed conversion did not differ between the control and SPAA kids. Mucosal protein concentrations were lower in the SPAA than the control kids. Concentrations of RNA and DNA did not differ between feeding groups, but in all kids mucosal RNA concentrations were higher in proximal than in middle and distal jejunum. Protein:RNA ratios were higher in the control than the SPAA kids and were lowest in proximal jejunum. Activities of alkaline phosphatase in enterocytes were higher in proximal than in middle and distal jejunum. Activities of mucosal xanthine oxidase were highest in distal jejunum and were higher in the SPAA than the control kids, especially in the middle and distal sites. The 15N-enrichment of mucosal RNA was higher in the control than the SPAA kids, especially in distal jejunum, and was lowest in distal jejunum. In contrast, 15N-enrichment of urea in plasma tended to be higher and Gly concentration in plasma was lower in the SPAA than the control kids. Data indicate that protein content and the protein:RNA ratio were lower in jejunal mucosa of goat kids fed milk replacer with partial replacement of CN protein by soy protein. These findings were accompanied by a lower level of reutilization of preformed dietary RNA precursors for RNA biosynthesis in jejunal mucosa and a higher activity of xanthine oxidase. Thus, feeding soy protein instead of CN protein reduced the incorporation of preformed dietary RNA precursors for RNA biosynthesis in the mucosa and activated key enzymes involved in nucleic acid breakdown.

Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar1[W][OA

PubMed Central

Voelker, Steven L.; Lachenbruch, Barbara; Meinzer, Frederick C.; Jourdes, Michael; Ki, Chanyoung; Patten, Ann M.; Davin, Laurence B.; Lewis, Norman G.; Tuskan, Gerald A.; Gunter, Lee; Decker, Stephen R.; Selig, Michael J.; Sykes, Robert; Himmel, Michael E.; Kitin, Peter; Shevchenko, Olga; Strauss, Steven H.

2010-01-01

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees. PMID:20729393

Dual Nature of Translational Control by Regulatory BC RNAs ▿

PubMed Central

Eom, Taesun; Berardi, Valerio; Zhong, Jun; Risuleo, Gianfranco; Tiedge, Henri

2011-01-01

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3′ stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems. PMID:21930783

RACK1 and the microRNA pathway: is it déjà-vu all over again?

PubMed

Speth, Corinna; Laubinger, Sascha

2014-01-01

MicroRNAs (miRNAs) control many aspects of development and adaption in plants and in animals by post-transcriptional control of mRNA stability and translatability. Over the last years numerous proteins have been identified in the miRNA pathway. The versatile scaffold protein RACK1 has been associated with efficient miRNA production and function in plants and metazoans. Here, we briefly summarize the differences of RACK1 function in the plant and animal miRNA pathways and discuss putative mechanisms and functional roles of RACK1 in miRNA biogenesis and action.

Metatranscriptomics of Soil Eukaryotic Communities.

PubMed

Yadav, Rajiv K; Bragalini, Claudia; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2016-01-01

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.

Tipping the balance of RNA stability by 3' editing of the transcriptome.

PubMed

Chung, Christina Z; Seidl, Lauren E; Mann, Mitchell R; Heinemann, Ilka U

2017-11-01

The regulation of active microRNAs (miRNAs) and maturation of messenger RNAs (mRNAs) that are competent for translation is a crucial point in the control of all cellular processes, with established roles in development and differentiation. Terminal nucleotidyltransferases (TNTases) are potent regulators of RNA metabolism. TNTases promote the addition of single or multiple nucleotides to an RNA transcript that can rapidly alter transcript stability. The well-known polyadenylation promotes transcript stability while the newly discovered but ubiquitious 3'-end polyuridylation marks RNA for degradation. Monoadenylation and uridylation are essential control mechanisms balancing mRNA and miRNA homeostasis. This review discusses the multiple functions of non-canonical TNTases, focusing on their substrate range, biological functions, and evolution. TNTases directly control mRNA and miRNA levels, with diverse roles in transcriptome stabilization, maturation, silencing, or degradation. We will summarize the current state of knowledge on non-canonical nucleotidyltransferases and their function in regulating miRNA and mRNA metabolism. We will review the discovery of uridylation as an RNA degradation pathway and discuss the evolution of nucleotidyltransferases along with their use in RNA labeling and future applications as therapeutic targets. The biochemically and evolutionarily highly related adenylyl- and uridylyltransferases play antagonizing roles in the cell. In general, RNA adenylation promotes stability, while uridylation marks RNA for degradation. Uridylyltransferases evolved from adenylyltransferases in multiple independent evolutionary events by the insertion of a histidine residue into the active site, altering nucleotide, but not RNA specificity. Understanding the mechanisms regulating RNA stability in the cell and controlling the transcriptome is essential for efforts aiming to influence cellular fate. Selectively enhancing or reducing RNA stability allows for alterations in the transcriptome, proteome, and downstream cellular processes. Genetic, biochemical, and clinical data suggest TNTases are potent targets for chemotherapeutics and have been exploited for RNA labeling applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

PubMed

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

Identification of suitable reference genes for hepatic microRNA quantitation.

PubMed

Lamba, Vishal; Ghodke-Puranik, Yogita; Guan, Weihua; Lamba, Jatinder K

2014-03-07

MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples. We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus, Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels. Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

Intratracheal administration of p38α short-hairpin RNA plasmid ameliorates lung ischemia-reperfusion injury in rats.

PubMed

Lv, Xiangqi; Tan, Jing; Liu, Dongdong; Wu, Ping; Cui, Xiaoguang

2012-06-01

Lung ischemia-reperfusion injury (LIRI) remains a significant problem after lung transplantation. A crucial signaling enzyme involved in inflammation and apoptosis during LIRI is p38 mitogen-activated protein kinase (MAPK). Gene silencing of p38α by short hairpin RNA (shRNA) can downregulate p38α expression. The lungs have an extremely large surface area, which makes the absorption of shRNA highly effective. Therefore, we evaluated the therapeutic efficacy of p38α shRNA plasmids in a rat model of lung transplantation. The delivery of p38α shRNA plasmid was performed by intratracheal administration 48 hours before transplantation into donor rats. Control animals received scrambled shRNA plasmids. Reverse-transcription polymerase chain reaction and Western blots were used to assess gene silencing efficacy. The therapeutic effects of shRNA plasmids were evaluated by lung function tests. We determined the levels of inflammatory cytokines, the level of intercellular adhesion molecule 1 (ICAM-1), c-Myc mRNA expression, and ICAM-1 protein expression, and the presence of cell apoptosis. Rats administered p38α shRNA plasmids showed a significant downregulation in lung expression of p38α transcripts and protein levels. Compared with the control group, the p38α shRNA group showed a higher pulmonary vein oxygen level, lower wet weight-to-dry weight ratio, lower lung injury score, and lower serum levels of tumor necrosis factor-α, interleukin-6, and interleukin-8. Messenger RNA levels of ICAM-1 and c-Myc in the p38α shRNA group were dramatically lower than in the control group. Levels of ICAM-1 protein expression exhibited a similar trend. Cell apoptosis decreased in the p38α shRNA group vs the control group. Intratracheal administration of p38α shRNA plasmids provided therapeutic effects in a rat model of lung transplantation. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

An oyster species-specific miRNA scaffold42648_5080 modulates haemocyte migration by targeting integrin pathway.

PubMed

Chen, Hao; Wang, Hao; Jiang, Shuai; Xu, Jiachao; Wang, Lingling; Qiu, Limei; Song, Linsheng

2016-10-01

miRNAs are important gene regulators at post-transcriptional level and can modulate diverse biological processes, including immune response. Dozens of species-specific miRNAs have been identified in oyster Crassostrea gigas while their functions remain largely unknown. In the present study, an oyster species-specific miRNA scaffold42648_5080 was found responsive to LPS stimulation and might target a total of 31 oyster genes possibly involved in cell communication, cellular localization and cellular response to stimulus. Besides, in gain-of-function assay of scaffold42648_5080 in vivo, the phagocytosis (30.90% in miRNA group verse 23.20% in miRNA control group), apoptosis (3.10% in miRNA group verse 5.30% in miRNA control group) and migration rate (13.88% in miRNA group verse 21.03% in miRNA control group) of oyster haemocytes were found significantly altered after the injection of scaffold42648_5080 mimics. Among the target genes, integrin-linked kinase (CgILK) was considered crucial in cell migration and its interaction with scaffold42648_5080 was then verified both in vitro and in vivo. Consequently, a significant decrease of relative luciferase ratio was observed in CgILK 3'-UTR luciferase reporter assay after transfection of scaffold42648_5080 mimics (0.70-fold of that in blank group, p < 0.01). Meanwhile, when scaffold42648_5080 was overexpressed in vivo (5.41-fold of miRNA control group, p < 0.01), the expression of CgILK declined significantly to 0.25-fold of miRNA control group (p < 0.01). Comparatively, a significant decrease of the haemocyte migration rate (19.76% verse 34.82% in siEGFP control group, p < 0.01) was observed after knock-down of CgILK in vivo. The present study, as far as we know, for the first time revealed the immunomodulation role of an oyster species-specific miRNA, which might provide new insights into miRNA-mediated adaptation mechanism of oysters. Copyright © 2016 Elsevier Ltd. All rights reserved.

The transcriptome of the developing grain: a resource for understanding seed development and the molecular control of the functional and nutritional properties of wheat.

PubMed

Rangan, Parimalan; Furtado, Agnelo; Henry, Robert J

2017-10-11

Wheat is one of the three major cereals that have been domesticated to feed human populations. The composition of the wheat grain determines the functional properties of wheat including milling efficiency, bread making, and nutritional value. Transcriptome analysis of the developing wheat grain provides key insights into the molecular basis for grain development and quality. The transcriptome of 35 genotypes was analysed by RNA-Seq at two development stages (14 and 30 days-post-anthesis, dpa) corresponding to the mid stage of development (stage Z75) and the almost mature seed (stage Z85). At 14dpa, most of the transcripts were associated with the synthesis of the major seed components including storage proteins and starch. At 30dpa, a diverse range of genes were expressed at low levels with a predominance of genes associated with seed defence and stress tolerance. RNA-Seq analysis of changes in expression between 14dpa and 30dpa stages revealed 26,477 transcripts that were significantly differentially expressed at a FDR corrected p-value cut-off at ≤0.01. Functional annotation and gene ontology mapping was performed and KEGG pathway mapping allowed grouping based upon biochemical linkages. This analysis demonstrated that photosynthesis associated with the pericarp was very active at 14dpa but had ceased by 30dpa. Recently reported genes for flour yield in milling and bread quality were found to influence wheat quality largely due to expression patterns at the earlier seed development stage. This study serves as a resource providing an overview of gene expression during wheat grain development at the early (14dpa) and late (30dpa) grain filling stages for use in studies of grain quality and nutritional value and in understanding seed biology.

Specific serum and CSF microRNA profiles distinguish sporadic behavioural variant of frontotemporal dementia compared with Alzheimer patients and cognitively healthy controls

PubMed Central

Oberhauser, Felix; Kornhuber, Johannes; Wiltfang, Jens; Fassbender, Klaus; Schroeter, Matthias L.; Volk, Alexander E.; Diehl-Schmid, Janine; Prudlo, Johannes; Danek, Adrian; Landwehrmeyer, Bernhard; Lauer, Martin; Otto, Markus; Jahn, Holger

2018-01-01

Information on circulating miRNAs in frontotemporal lobar degeneration is very limited and conflicting results have complicated an interpretation in Alzheimer’s disease thus far. In the present study we I) collected samples from multiple clinical centers across Germany, II) defined 3 homogenous patient groups with high sample sizes (bvFTD n = 48, AD n = 48 and cognitively healthy controls n = 44), III) compared expression levels in both CSF and serum samples and IV) detected a limited set of miRNAs by using a MIQE compliant protocol based on SYBR-green miRCURY assays that have proven reliable to generate reproducible results. We included several quality controls that identified and reduced technical variation to increase the reliability of our data. We showed that the expression levels of circulating miRNAs measured in CSF did not correlate with levels in serum. Using cluster analysis we found expression pattern in serum that, in part, reflects the genomic organization and affiliation to a specific miRNA family and that were specifically altered in bvFTD, AD, and control groups. Applying factor analysis we identified a 3-factor model characterized by a miRNA signature that explained 80% of the variance classifying healthy controls with 97%, bvFTD with 77% and AD with 72% accuracy. MANOVA confirmed signals like miR-320a and miR-26b-5p at BH corrected significance that contributed most to discriminate bvFTD cases with 96% sensitivity and 90% specificity and AD cases with 89% sensitivity and specificity compared to healthy controls, respectively. Correlation analysis revealed that miRNAs from the 3-factor model also correlated with levels of protein biomarker amyloid-beta1-42 and phosphorylated neurofilament heavy chain, indicating their potential role in the monitoring of progressive neuronal degeneration. Our data show that miRNAs can be reproducibly measured in serum and CSF without pre-amplification and that serum includes higher expressed signals that demonstrate an overall better ability to classify bvFTD, AD and healthy controls compared to signals detected in CSF. PMID:29746584

NASA's Rodent Research Project: Validation of Flight Hardware, Operations and Science Capabilities for Conducting Long Duration Experiments in Space

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Beegle, J. E.; Wigley, C. L.; Pletcher, D.; Globus, R. K.

2015-01-01

Research using rodents is an essential tool for advancing biomedical research on Earth and in space. Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed at the NASA Ames Research Center. Twenty C57BL/6J adult female mice were launched on Sept 21, 2014 in a Dragon Capsule (SpaceX-4), then transferred to the ISS for a total time of 21-22 days (10 commercial mice) or 37 (10 validation mice). Tissues collected on-orbit were either rapidly frozen or preserved in RNA later at less than or equal to -80 C (n=2/group) until their return to Earth. Remaining carcasses were rapidly frozen for dissection post-flight. The three controls groups at Kennedy Space Center consisted of: Basal mice euthanized at the time of launch, Vivarium controls, housed in standard cages, and Ground Controls (GC), housed in flight hardware within an environmental chamber. FLT mice appeared more physically active on-orbit than GC, and behavior analysis are in progress. Upon return to Earth, there were no differences in body weights between FLT and GC at the end of the 37 days in space. RNA was of high quality (RIN greater than 8.5). Liver enzyme activity levels of FLT mice and all control mice were similar in magnitude to those of the samples that were optimally processed in the laboratory. Liver samples collected from the intact frozen FLT carcasses had RNA RIN of 7.27 +/- 0.52, which was lower than that of the samples processed on-orbit, but similar to those obtained from the control group intact carcasses. Nonetheless, the RNA samples from the intact carcasses were acceptable for the most demanding transcriptomic analyses. Adrenal glands, thymus and spleen (organs associated with stress response) showed no significant difference in weights between FLT and GC. Enzymatic activity was also not significantly different. Over 3,000 tissues collected from the four groups of mice have become available for the Biospecimen Sharing Program. Together, these validation flight findings demonstrate the capability to support long-duration RR on the ISS to achieve both basic science and biomedical objectives.

RNA-Seq for Bacterial Gene Expression.

PubMed

Poulsen, Line Dahl; Vinther, Jeppe

2018-06-01

RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo.

PubMed

Su, Jianguo; Zhu, Zuoyan; Wang, Yaping; Xiong, Feng; Zou, Jun

2008-01-01

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Enzymes Involved in Post-transcriptional RNA Metabolism in Gram-negative bacteria

PubMed Central

Mohanty, Bijoy K.

2018-01-01

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways. PMID:29676246

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules.

PubMed

Kobayashi, Ryo; Biyani, Manish; Ueno, Shingo; Kumal, Subhashini Raj; Kuramochi, Hiromi; Ichiki, Takanori

2015-05-15

We have developed an advanced microintaglio printing method for fabricating fine and high-density micropatterns and applied it to the microarraying of RNA molecules. The microintaglio printing of RNA reported here is based on the hybridization of RNA with immobilized complementary DNA probes. The hybridization was controlled by switching the RNA conformation via the temperature, and an RNA microarray with a diameter of 1.5 µm and a density of 40,000 spots/mm(2) with high contrast was successfully fabricated. Specifically, no size effects were observed in the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude. Additionally, we have developed a microintaglio printing method for transcribed RNA microarrays on demand using DNA-immobilized magnetic beads. The beads were arrayed on wells fabricated on a printing mold and the wells were filled with in vitro transcription reagent and sealed with a DNA-immobilized glass substrate. Subsequently, RNA was in situ synthesized using the bead-immobilized DNA as a template and printed onto the substrate via hybridization. Since the microintaglio printing of RNA using DNA-immobilized beads enables the fabrication of a microarray of spots composed of multiple RNA sequences, it will be possible to screen or analyze RNA functions using an RNA microarray fabricated by temperature-controlled microintaglio printing (TC-µIP). Copyright © 2014 Elsevier B.V. All rights reserved.

Context-dependent control of alternative splicing by RNA-binding proteins

PubMed Central

Fu, Xiang-Dong; Ares, Manuel

2015-01-01

Sequence-specific RNA-binding proteins (RBPs) bind to pre-mRNA to control alternative splicing, but it is not yet possible to read the ‘splicing code’ that dictates splicing regulation on the basis of genome sequence. Each alternative splicing event is controlled by multiple RBPs, the combined action of which creates a distribution of alternatively spliced products in a given cell type. As each cell type expresses a distinct array of RBPs, the interpretation of regulatory information on a given RNA target is exceedingly dependent on the cell type. RBPs also control each other’s functions at many levels, including by mutual modulation of their binding activities on specific regulatory RNA elements. In this Review, we describe some of the emerging rules that govern the highly context-dependent and combinatorial nature of alternative splicing regulation. PMID:25112293

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

PubMed

Kopechek, Jonathan A; Carson, Andrew R; McTiernan, Charles F; Chen, Xucai; Klein, Edwin C; Villanueva, Flordeliza S

2016-01-01

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals

PubMed Central

Atanasov, Janina; Groher, Florian

2017-01-01

Abstract Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis. PMID:29036355

Approaches for Investigating Translational Regulation Controlled by PARP1: Biotin-Based UV Cross-Linking and Luciferase Reporter Assay.

PubMed

Ji, Yingbiao

2017-01-01

The RNA-binding proteins (RBPs) play a pivotal role in controlling gene expression through posttranscriptional processes. As the trans-acting factors, RBPs interact with the cis-regulatory elements located within mRNAs to regulate mRNA translational efficiency. Adding a new-layer regulation, recent studies suggest that poly(ADP-ribosyl)ation of the RNA-binding proteins often inhibit the RNA-binding ability of RBPs, thus regulating RBP-dependent mRNA metabolism including translational control. Here, we describe a biotin-based UV cross-linking method to determine if excessive accumulation of pADPr in the cell disrupts the interaction between RBPs and their target mRNAs. In addition, we illustrate the protocol of using the luciferase reporter assay to determine the effect of poly(ADP-ribosyl)ation on mRNA translation.

A practical tissue sampling method using ordinary paper for molecular detection of infectious bursal disease virus RNA by RT-PCR.

PubMed

Maw, Min Thein; Yamaguchi, Tsuyoshi; Kasanga, Christopher J; Terasaki, Kaori; Fukushi, Hideto

2006-12-01

A practical sampling method for bursal tissue using ordinary paper for molecular diagnosis of infectious bursal disease (IBD) was established. IBD virus-infected bursa was directly smeared on chromatography paper, filter paper, or stationery copy paper and was then fixed with absolute ethanol, Tris-HCl-saturated phenol, or phenol:chloroform:isoamyl alcohol (25:24:1). Flinders Technology Associates (FTA) card, which is designed for the collection of biological samples for molecular detection, was also used. After storage at 37 C for up to 30 days, total RNA directly extracted from the tissue fixed on the papers and FTA card were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of IBD virus (IBDV) RNA. In addition, the ability of each chemical used in the fixation and the FTA card to inactivate IBDV was evaluated. Regardless of the paper quality, storage period, and fixation method, IBDV RNA was consistently detected in all of the samples. IBDV in the bursal tissue was inactivated with phenol but not with ethanol or the unknown chemicals in FTA card. These results show that ordinary papers sustain the viral RNA, as does FTA card, but phenol fixation is superior to FTA card in inactivating IBDV. The new sampling method using ordinary paper with phenol fixation is safe, inexpensive, simple, and easy, and is thus suitable for conducting a global survey of IBD even where laboratory resources are limited. This practical method should contribute to the control of IBD worldwide.

Visualization of RNA–protein interactions in living cells: FMRP and IMP1 interact on mRNAs

PubMed Central

Rackham, Oliver; Brown, Chris M

2004-01-01

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA–protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells. PMID:15282548

Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish.

PubMed

Bouleau, Aurélien; Desvignes, Thomas; Traverso, Juan Martin; Nguyen, Thaovi; Chesnel, Franck; Fauvel, Christian; Bobe, Julien

2014-08-01

The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates. © 2014 by the Society for the Study of Reproduction, Inc.

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a

PubMed Central

Jeyapalan, Jessie C.; Sedivy, John M.

2013-01-01

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5×107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems. PMID:23454889

How to measure RNA expression in rare senescent cells expressing any specific protein such as p16Ink4a.

PubMed

Jeyapalan, Jessie C; Sedivy, John M

2013-02-01

Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5x107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.

Microaspiration of Solanum tuberosum root cells at early stages of infection by Globodera pallida.

PubMed

Kooliyottil, Rinu; Dandurand, Louise-Marie; Kuhl, Joseph C; Caplan, Allan; Xiao, Fangming

2017-01-01

Sedentary endoparasitic cyst nematodes form a feeding structure in plant roots, called a syncytium. Syncytium formation involves extensive transcriptional modifications, which leads to cell modifications such as increased cytoplasmic streaming, enlarged nuclei, increased numbers of organelles, and replacement of a central vacuole by many small vacuoles. When whole root RNA is isolated and analyzed, transcript changes manifested in the infected plant cells are overshadowed by gene expression from cells of the entire root system. Use of microaspiration allows isolation of the content of nematode infected cells from a heterogeneous cell population. However, one challenge with this method is identifying the nematode infected cells under the microscope at early stages of infection. This problem was addressed by staining nematode juveniles with a fluorescent dye prior to infection so that the infected cells could be located and microaspirated. In the present study, we used the fluorescent vital stain PKH26 coupled with a micro-rhizosphere chamber to locate the infected nematode Globodera pallida in Solanum tuberosum root cells. This enabled microaspiration of nematode-infected root cells during the early stages of parasitism. To study the transcriptional events occurring in these cells, an RNA isolation method from microaspirated samples was optimized, and subsequently the RNA was purified using magnetic beads. With this method, we obtained an RNA quality number of 7.8. For transcriptome studies, cDNA was synthesized from the isolated RNA and assessed by successfully amplifying several pathogenesis related protein coding genes. The use of PKH26 stained nematode juveniles enabled early detection of nematode infected cells for microaspiration. To investigate transcriptional changes in low yielding RNA samples, bead-based RNA extraction procedures minimized RNA degradation and provided high quality RNA. This protocol provides a robust procedure to analyze gene expression in nematode-infected cells.

An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep

PubMed Central

Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

2016-01-01

Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep’s wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium waves included 13 downregulated miRNAs that had regulatory effects on 64 upregulated genes and 4 upregulated miRNAs, which in turn had regulatory effects on 22 downregulated genes. Compared to medium waves, large waves consisted of 13 upregulated miRNAs that had regulatory effects on 48 downregulated genes. These differentially expressed miRNAs and genes may play a significant role in forming different patterns, and provide evidence for the molecular mechanisms underlying the formation of hair follicles of varying patterns. PMID:27404636

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid

PubMed Central

2012-01-01

Background Jatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission. Results The enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta 12 fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants. Conclusion In this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil. PMID:22377043

Modelling the CDK-dependent transcription cycle in fission yeast.

PubMed

Sansó, Miriam; Fisher, Robert P

2013-12-01

CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.

Human health and ecological risk assessments for SmartStax PRO (MON 89034 x TC1507 x MON 87411 x DAS-59122-7), a plant-incorporated protectant intended to control corn rootworm through ribonucleic acid (RNA) interference

EPA Science Inventory

The use of RNA interference (RNAi) gene silencing technology, particularly RNAi for pesticidal purposes to control macroorganism pests, is a relatively recent innovation. Post-transcriptional silencing of gene function is a very rapid process where double-stranded RNA (dsRNA) dir...

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Protein Kinase A Regulates Molecular Chaperone Transcription and Protein Aggregation

PubMed Central

Prince, Thomas; Calderwood, Stuart K.

2011-01-01

Heat shock factor 1 (HSF1) regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington's disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAcα) and becomes phosphorylated on at least one regulatory serine residue (S320). We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb) and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q) tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAcα, or the pTEFb component CDK9, indicating a key role for the HSF1-PKA cascade in protein quality control. PMID:22216146

5SRNAdb: an information resource for 5S ribosomal RNAs.

PubMed

Szymanski, Maciej; Zielezinski, Andrzej; Barciszewski, Jan; Erdmann, Volker A; Karlowski, Wojciech M

2016-01-04

Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

PubMed

Reid-Bayliss, Kate S; Loeb, Lawrence A

2017-08-29

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

RNA Bricks—a database of RNA 3D motifs and their interactions

PubMed Central

Chojnowski, Grzegorz; Waleń, Tomasz; Bujnicki, Janusz M.

2014-01-01

The RNA Bricks database (http://iimcb.genesilico.pl/rnabricks), stores information about recurrent RNA 3D motifs and their interactions, found in experimentally determined RNA structures and in RNA–protein complexes. In contrast to other similar tools (RNA 3D Motif Atlas, RNA Frabase, Rloom) RNA motifs, i.e. ‘RNA bricks’ are presented in the molecular environment, in which they were determined, including RNA, protein, metal ions, water molecules and ligands. All nucleotide residues in RNA bricks are annotated with structural quality scores that describe real-space correlation coefficients with the electron density data (if available), backbone geometry and possible steric conflicts, which can be used to identify poorly modeled residues. The database is also equipped with an algorithm for 3D motif search and comparison. The algorithm compares spatial positions of backbone atoms of the user-provided query structure and of stored RNA motifs, without relying on sequence or secondary structure information. This enables the identification of local structural similarities among evolutionarily related and unrelated RNA molecules. Besides, the search utility enables searching ‘RNA bricks’ according to sequence similarity, and makes it possible to identify motifs with modified ribonucleotide residues at specific positions. PMID:24220091

mRNA of cytokines in bone marrow and bone biomarkers in response to propranolol in a nutritional growth retardation model.

PubMed

Tasat, Deborah R; Lezón, Christian E; Astort, Francisco; Pintos, Patricia M; Macri, Elisa V; Friedman, Silvia M; Boyer, Patricia M

2014-10-01

The aim of this study was to assess mRNA of IL-6, TNFα and IL-10 cytokines in bone marrow, possible mediators involved in altered bone remodeling with detrimental consequences on bone quality in NGR (Nutritional growth retardation) rats. Weanling male Wistar rats were assigned either to control (C) or experimental group (NGR) (n=20 each). C and NGR groups were assigned to 2 groups according to receiving saline solution (SS) or propranolol hydrochloride (P): C, C+P (CP), NGR or NGR+P (NGRP). For 4 weeks, NGR and NGRP rats received 80% of the amount of food consumed by C and CP, respectively, the previous day, corrected by body weight. P (7 mg/kg/day) was injected ip 5 days/week, for 4 weeks in CP and NGRP rats. Body weight and length were recorded. After 4 weeks, blood was drawn. Femurs were dissected for RNA isolation from bone marrow and mRNA of cytokines assays. Food restriction induced a significant negative effect on body growth in NGR and NGRP rats (p<0.001). P had no effects on zoometric parameters (p>0.05). CTX-I increased in NGR rats vs. C (p<0.001), but diminished in NGRP (p<0.01). Serum osteocalcin, PTH, calcium and phosphate levels remained unchanged between groups (p>0.05). In NGR, bone marrow IL-6 mRNA and IL-10 mRNA levels were low as compared to other groups (p<0.05). In contrast, bone marrow TNF-α mRNA levels were significantly high (p<0.05). This study provides evidences that NGR outcomes in a bone marrow proinflammatory microenvironment leading to unbalanced bone remodeling by enhancement of bone resorption reverted by propranolol. Copyright © 2014. Published by Elsevier Urban & Partner Sp. z o.o.

Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp

PubMed Central

Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

2016-01-01

In mammals, body temperature fluctuates diurnally around a mean value of 36°C–37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells’ circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide “approach to steady-state” kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

Development of a quantitative assay to measure expression of transforming growth factor ß (TGF-ß) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

USGS Publications Warehouse

Robertson, Laura S.; Ottinger, Christopher A.; Burdick, Summer M.; VanderKooi, Scott P.

2012-01-01

The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.

Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment

PubMed Central

Dasari, Surendra; Chambers, Matthew C.; Martinez, Misti A.; Carpenter, Kristin L.; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo J.; Tabb, David L.

2012-01-01

Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines. PMID:22217208

[Lentiviral vector-mediated short hairpin RNA targeting survivin inhibits abdominal growth of human endometrium xenograft in nude mice].

PubMed

Peng, Dongxian; He, Yuanli

2015-02-01

To investigate the inhibitory effect of lentiviral vector-mediated short hairpin RNA targeting survivin (LV-survivin shRNA) on the growth of human endometrium xenograft in the abdominal cavity of nude mice. The endometrium xenografts from 8 women with endometriosis were injected into the peritoneal cavities of 45 nude mice. The mice were then randomly assigned to receive intraperitoneal injection of LV-survivin shRNA, pGCL-NC-GFP (negative control) or PBS (blank control). Two weeks later, the number and morphometry of endometriotic lesions were quantified and the expression of survivin protein were detected by immunohistochemistry. The formation of endometriotic lesions was significantly suppressed in mice receiving LV-survivin shRNA injection as compared with those in the two control groups (P/0.001). The mice in LV-survivin-shRNA group showed significantly down-regulated expression levels of survivin protein compared with those in the negative and blank control groups, presenting also necrosis in the endometriosis-like lesions in microscopic observation. Lentiviral vector-mediated shRNA can effectively inhibit the expression of survivin in human endometrium xengrafts and suppress the formation and growth of endometriotic lesions in the abdominal cavities of nude mice.

The complete mitochondrial genome of Chrysopa pallens (Insecta, Neuroptera, Chrysopidae).

PubMed

He, Kun; Chen, Zhe; Yu, Dan-Na; Zhang, Jia-Yong

2012-10-01

The complete mitochondrial genome of Chrysopa pallens (Neuroptera, Chrysopidae) was sequenced. It consists of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA (rRNA) genes, and a control region (AT-rich region). The total length of C. pallens mitogenome is 16,723 bp with 79.5% AT content, and the length of control region is 1905 bp with 89.1% AT content. The non-coding regions of C. pallens include control region between 12S rRNA and trnI genes, and a 75-bp space region between trnI and trnQ genes.

A long antisense RNA in plant chloroplasts.

PubMed

Georg, J; Honsel, A; Voss, B; Rennenberg, H; Hess, W R

2010-05-01

Based on computational prediction of RNA secondary structures, a long antisense RNA (asRNA) was found in chloroplasts of Arabidopsis, Nicotiana tabacum and poplar, which occurs in two to three major transcripts. Mapping of primary 5' ends, northern hybridizations and quantitative real-time reverse transcription polymerase chain reaction (qPCR) experiments demonstrated that these transcripts originate from a promoter that is typical for the plastid-encoded RNA polymerase and are over their full length in antisense orientation to the gene ndhB and therefore were designated asRNA_ndhB. The asRNA_ndhB transcripts predominantly accumulate in young leaves and at physiological growth temperatures. Two nucleotide positions in the mRNA that are subject to C-to-U RNA editing and which were previously found to be sensitive to elevated temperatures are covered by asRNA_ndhB. Nevertheless, the correlation between the accumulation of asRNA_ndhB and RNA editing appeared weak in a temperature shift experiment. With asRNA_ndhB, we describe the first asRNA of plant chloroplasts that covers RNA editing sites, as well as a group II intron splice acceptor site, and that is under developmental control, raising the possibility that long asRNAs could be involved in RNA maturation or the control of RNA stability.

Determination of the conformational ensemble of the TAR RNA by X-ray scattering interferometry

PubMed Central

Walker, Peter

2017-01-01

Abstract The conformational ensembles of structured RNA's are crucial for biological function, but they remain difficult to elucidate experimentally. We demonstrate with HIV-1 TAR RNA that X-ray scattering interferometry (XSI) can be used to determine RNA conformational ensembles. X-ray scattering interferometry (XSI) is based on site-specifically labeling RNA with pairs of heavy atom probes, and precisely measuring the distribution of inter-probe distances that arise from a heterogeneous mixture of RNA solution structures. We show that the XSI-based model of the TAR RNA ensemble closely resembles an independent model derived from NMR-RDC data. Further, we show how the TAR RNA ensemble changes shape at different salt concentrations. Finally, we demonstrate that a single hybrid model of the TAR RNA ensemble simultaneously fits both the XSI and NMR-RDC data set and show that XSI can be combined with NMR-RDC to further improve the quality of the determined ensemble. The results suggest that XSI-RNA will be a powerful approach for characterizing the solution conformational ensembles of RNAs and RNA-protein complexes under diverse solution conditions. PMID:28108663

Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.

PubMed

Sandstedt, Mikael; Jonsson, Marianne; Asp, Julia; Dellgren, Göran; Lindahl, Anders; Jeppsson, Anders; Sandstedt, Joakim

2015-12-01

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.

Metabolic Reprogramming in Leaf Lettuce Grown Under Different Light Quality and Intensity Conditions Using Narrow-Band LEDs.

PubMed

Kitazaki, Kazuyoshi; Fukushima, Atsushi; Nakabayashi, Ryo; Okazaki, Yozo; Kobayashi, Makoto; Mori, Tetsuya; Nishizawa, Tomoko; Reyes-Chin-Wo, Sebastian; Michelmore, Richard W; Saito, Kazuki; Shoji, Kazuhiro; Kusano, Miyako

2018-05-21

Light-emitting diodes (LEDs) are an artificial light source used in closed-type plant factories and provide a promising solution for a year-round supply of green leafy vegetables, such as lettuce (Lactuca sativa L.). Obtaining high-quality seedlings using controlled irradiation from LEDs is critical, as the seedling health affects the growth and yield of leaf lettuce after transplantation. Because key molecular pathways underlying plant responses to a specific light quality and intensity remain poorly characterised, we used a multi-omics-based approach to evaluate the metabolic and transcriptional reprogramming of leaf lettuce seedlings grown under narrow-band LED lighting. Four types of monochromatic LEDs (one blue, two green and one red) and white fluorescent light (control) were used at low and high intensities (100 and 300 μmol·m -2 ·s -1 , respectively). Multi-platform mass spectrometry-based metabolomics and RNA-Seq were used to determine changes in the metabolome and transcriptome of lettuce plants in response to different light qualities and intensities. Metabolic pathway analysis revealed distinct regulatory mechanisms involved in flavonoid and phenylpropanoid biosynthetic pathways under blue and green wavelengths. Taken together, these data suggest that the energy transmitted by green light is effective in creating a balance between biomass production and the production of secondary metabolites involved in plant defence.

Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-fixed paraffin-embedded (FFPE) Samples

EPA Science Inventory

Formalin-fixed paraffin-embedded (FFPE) samples provide a vast untapped resource for chemical safety and translational science. To date, genomic profiling of FFPE samples has been limited by poor RNA quality and inconsistent results with limited utility in dose-response assessmen...

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound

PubMed Central

McTiernan, Charles F.; Chen, Xucai; Klein, Edwin C.; Villanueva, Flordeliza S.

2016-01-01

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

A View on the Importance of "Multi-Attribute Method" for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy.

PubMed

Rogers, Richard S; Abernathy, Michael; Richardson, Douglas D; Rouse, Jason C; Sperry, Justin B; Swann, Patrick; Wypych, Jette; Yu, Christopher; Zang, Li; Deshpande, Rohini

2017-11-30

Today, we are experiencing unprecedented growth and innovation within the pharmaceutical industry. Established protein therapeutic modalities, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, are being used to treat previously unmet medical needs. Novel therapies such as bispecific T cell engagers (BiTEs), chimeric antigen T cell receptors (CARTs), siRNA, and gene therapies are paving the path towards increasingly personalized medicine. This advancement of new indications and therapeutic modalities is paralleled by development of new analytical technologies and methods that provide enhanced information content in a more efficient manner. Recently, a liquid chromatography-mass spectrometry (LC-MS) multi-attribute method (MAM) has been developed and designed for improved simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes (Rogers et al. MAbs 7(5):881-90, 2015). Based on peptide mapping principles, this powerful tool represents a true advancement in testing methodology that can be utilized not only during product characterization, formulation development, stability testing, and development of the manufacturing process, but also as a platform quality control method in dispositioning clinical materials for both innovative biotherapeutics and biosimilars.

Comparative genomics of metabolic capacities of regulons controlled by cis-regulatory RNA motifs in bacteria.

PubMed

Sun, Eric I; Leyn, Semen A; Kazanov, Marat D; Saier, Milton H; Novichkov, Pavel S; Rodionov, Dmitry A

2013-09-02

In silico comparative genomics approaches have been efficiently used for functional prediction and reconstruction of metabolic and regulatory networks. Riboswitches are metabolite-sensing structures often found in bacterial mRNA leaders controlling gene expression on transcriptional or translational levels.An increasing number of riboswitches and other cis-regulatory RNAs have been recently classified into numerous RNA families in the Rfam database. High conservation of these RNA motifs provides a unique advantage for their genomic identification and comparative analysis. A comparative genomics approach implemented in the RegPredict tool was used for reconstruction and functional annotation of regulons controlled by RNAs from 43 Rfam families in diverse taxonomic groups of Bacteria. The inferred regulons include ~5200 cis-regulatory RNAs and more than 12000 target genes in 255 microbial genomes. All predicted RNA-regulated genes were classified into specific and overall functional categories. Analysis of taxonomic distribution of these categories allowed us to establish major functional preferences for each analyzed cis-regulatory RNA motif family. Overall, most RNA motif regulons showed predictable functional content in accordance with their experimentally established effector ligands. Our results suggest that some RNA motifs (including thiamin pyrophosphate and cobalamin riboswitches that control the cofactor metabolism) are widespread and likely originated from the last common ancestor of all bacteria. However, many more analyzed RNA motifs are restricted to a narrow taxonomic group of bacteria and likely represent more recent evolutionary innovations. The reconstructed regulatory networks for major known RNA motifs substantially expand the existing knowledge of transcriptional regulation in bacteria. The inferred regulons can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis. The obtained genome-wide collection of reference RNA motif regulons is available in the RegPrecise database (http://regprecise.lbl.gov/).

The Effect of Different Pollination on the Expression of Dangshan Su Pear MicroRNA

PubMed Central

Cheng, Xi; Yan, Chongchong; Zhang, Jinyun; Ma, Chenhui; Li, Shumei; Jin, Qing; Zhang, Nan; Cao, Yunpeng; Lin, Yi

2017-01-01

The high-throughput sequencing of pear “Dangshan Su” × “Yali” (whose fruits lignin and stone cell content are high and quality is poor) and pear “Dangshan Su” × “Wonhwang” (whose fruits with low content of lignin and stone cell and the quality are better ) found that the expressions of these two miRNAs (pyr-1809 and pyr-novel-miR-144-3p) were significantly different; their corresponding target genes encode two kinds of laccase (Pbr018935.1 and Pbr003857.1). qRT-PCR results showed that these two enzymes are involved in the formation of lignin and stone cells and the existence of these two miRNAs has a negative effect on them. It was concluded that the effect of pollination on the development of stone cells may affect the synthesis of lignin, through the regulation of laccase controlled by miRNAs, and ultimately affect the formation of stone cell and fruit quality. PMID:28497043

The 7SK snRNP associates with the little elongation complex to promote snRNA gene expression.

PubMed

Egloff, Sylvain; Vitali, Patrice; Tellier, Michael; Raffel, Raoul; Murphy, Shona; Kiss, Tamás

2017-04-03

The 7SK small nuclear RNP (snRNP), composed of the 7SK small nuclear RNA (snRNA), MePCE, and Larp7, regulates the mRNA elongation capacity of RNA polymerase II (RNAPII) through controlling the nuclear activity of positive transcription elongation factor b (P-TEFb). Here, we demonstrate that the human 7SK snRNP also functions as a canonical transcription factor that, in collaboration with the little elongation complex (LEC) comprising ELL, Ice1, Ice2, and ZC3H8, promotes transcription of RNAPII-specific spliceosomal snRNA and small nucleolar RNA (snoRNA) genes. The 7SK snRNA specifically associates with a fraction of RNAPII hyperphosphorylated at Ser5 and Ser7, which is a hallmark of RNAPII engaged in snRNA synthesis. Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) experiments revealed enrichments for all components of the 7SK snRNP on RNAPII-specific sn/snoRNA genes. Depletion of 7SK snRNA or Larp7 disrupts LEC integrity, inhibits RNAPII recruitment to RNAPII-specific sn/snoRNA genes, and reduces nascent snRNA and snoRNA synthesis. Thus, through controlling both mRNA elongation and sn/snoRNA synthesis, the 7SK snRNP is a key regulator of nuclear RNA production by RNAPII. © 2017 The Authors.

Assembly of RNA nanostructures on supported lipid bilayers

PubMed Central

Dabkowska, Aleksandra P.; Michanek, Agnes; Jaeger, Luc; Rabe, Michael; Chworos, Arkadiusz; Höök, Fredrik; Nylander, Tommy; Sparr, Emma

2014-01-01

The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nanostructures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces. PMID:25417592

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences.

PubMed

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccas e 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like ( Sil1 ), scavenger receptor class C ( Src ), and scavenger receptor class B ( Srb3 and Srb4 ) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences

PubMed Central

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccase 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like (Sil1), scavenger receptor class C (Src), and scavenger receptor class B (Srb3 and Srb4) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean. PMID:29773992

Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

PubMed

Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

2018-03-16

Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing.

PubMed

Sachsenröder, Jana; Twardziok, Sven; Hammerl, Jens A; Janczyk, Pawel; Wrede, Paul; Hertwig, Stefan; Johne, Reimar

2012-01-01

Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pig virus. The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures comparability of the method and may be used for further method optimization.

An Improved Total RNA Extraction Method for White Jelly Mushroom Tremella fuciformis Rich in Polysaccharides

PubMed Central

Zhu, Hanyu; Sun, Xueyan; Liu, Dongmei; Zheng, Liesheng; Chen, Liguo

2017-01-01

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 µg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides. PMID:29371814

RNA extraction from various recalcitrant plant tissues with a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment.

PubMed

Suzuki, Yuji; Mae, Tadahiko; Makino, Amane

2008-07-01

High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction.

Preservation of RNA and DNA from mammal samples under field conditions.

PubMed

Camacho-Sanchez, Miguel; Burraco, Pablo; Gomez-Mestre, Ivan; Leonard, Jennifer A

2013-07-01

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months. © 2013 John Wiley & Sons Ltd.

Puromycin-resistant lentiviral control shRNA vector, pLKO.1 induces unexpected cellular differentiation of P19 embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanungo, Jyotshna

RNA silencing is used as a common method for investigating loss-of-function effects of genes of interest. In mammalian cells, RNA interference (RNAi) or RNA silencing can be achieved by transient siRNA (small or short interfering RNA) transfection or by stable shRNA (short hairpin RNA) systems. Various vectors are used for efficient delivery of shRNA. Lentiviral vectors offer an efficient delivery system for stable and long-term expression of the shRNA in mammalian cells. The widely used lentiviral pLKO.1 plasmid vector is very popular in RNAi studies. A large RNAi database, a TRC (the RNAi Consortium) library, was established based on themore » pLKO.1-TRC plasmid vector. This plasmid (also called pLKO.1-puro) has a puromycin-resistant gene for selection in mammalian cells along with designs for generating lentiviral particles as well for RNA silencing. While using the pLKO.1-puro TRC control shRNA plasmid for transfection in murine P19 embryonic stem (ES) cells, it was unexpectedly discovered that this plasmid vector induced robust endodermal differentiation. Since P19 ES cells are pluripotent and respond to external stimuli that have the potential to alter the phenotype and thus its stemness, other cell types used in RNA silencing studies do not display the obvious effect and therefore, may affect experiments in subtle ways that would go undetected. This study for the first time provides evidence that raises concern and warrants extreme caution while using the pLKO.1-puro control shRNA vector because of its unexpected non-specific effects on cellular integrity. - Highlights: • In P19 ES cells the pLKO.1-puro lentiviral control shRNA vector induced endodermal differentiation. • P19 ES cells harboring the pCDNA3 plasmid vector retained their stem-ness as opposed to those harboring the pLKO.1-puro vector. • P19 ES cells can serve as a sensor to determine vector safety. • Extreme caution is warranted while using the widely used pLKO.1-puro lentiviral vector for experimental and therapeutic designs.« less

miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.

PubMed

Martínez, Cristina; Rodiño-Janeiro, Bruno K; Lobo, Beatriz; Stanifer, Megan L; Klaus, Bernd; Granzow, Martin; González-Castro, Ana M; Salvo-Romero, Eloisa; Alonso-Cotoner, Carmen; Pigrau, Marc; Roeth, Ralph; Rappold, Gudrun; Huber, Wolfgang; González-Silos, Rosa; Lorenzo, Justo; de Torres, Inés; Azpiroz, Fernando; Boulant, Steeve; Vicario, María; Niesler, Beate; Santos, Javier

2017-09-01

Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs. Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells. IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins. Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®

PubMed Central

Golubeva, Yelena G.; Smith, Roberta M.; Sternberg, Lawrence R.

2013-01-01

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues. PMID:23805281

Conferring high-temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene.

PubMed

Nakamura, Shinya; Hondo, Kana; Kawara, Tomoko; Okazaki, Yozo; Saito, Kazuki; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Nishiguchi, Masamichi

2016-02-01

We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Comparative Genomics as a Foundation for Evo-Devo Studies in Birds.

PubMed

Grayson, Phil; Sin, Simon Y W; Sackton, Timothy B; Edwards, Scott V

2017-01-01

Developmental genomics is a rapidly growing field, and high-quality genomes are a useful foundation for comparative developmental studies. A high-quality genome forms an essential reference onto which the data from numerous assays and experiments, including ChIP-seq, ATAC-seq, and RNA-seq, can be mapped. A genome also streamlines and simplifies the development of primers used to amplify putative regulatory regions for enhancer screens, cDNA probes for in situ hybridization, microRNAs (miRNAs) or short hairpin RNAs (shRNA) for RNA interference (RNAi) knockdowns, mRNAs for misexpression studies, and even guide RNAs (gRNAs) for CRISPR knockouts. Finally, much can be gleaned from comparative genomics alone, including the identification of highly conserved putative regulatory regions. This chapter provides an overview of laboratory and bioinformatics protocols for DNA extraction, library preparation, library quantification, and genome assembly, from fresh or frozen tissue to a draft avian genome. Generating a high-quality draft genome can provide a developmental research group with excellent resources for their study organism, opening the doors to many additional assays and experiments.

Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage.

PubMed

Yang, Lijun; Cui, Hong; Cao, Ting

2014-03-01

Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair. miRNA-9 is involved in the occurrence of many related neurological disorders. Bioinformatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups: control group; oxygen-glucose deprivation group (treatment with 8% O2 + 92% N2 and sugar-free medium for 60 minutes); transfection control group (after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid) and miRNA-9 transfection group (after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid). From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligodendrocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.

Natural antisense RNAs as mRNA regulatory elements in bacteria: a review on function and applications.

PubMed

Saberi, Fatemeh; Kamali, Mehdi; Najafi, Ali; Yazdanparast, Alavieh; Moghaddam, Mehrdad Moosazadeh

2016-01-01

Naturally occurring antisense RNAs are small, diffusible, untranslated transcripts that pair to target RNAs at specific regions of complementarity to control their biological function by regulating gene expression at the post-transcriptional level. This review focuses on known cases of antisense RNA control in prokaryotes and provides an overview of some natural RNA-based mechanisms that bacteria use to modulate gene expression, such as mRNA sensors, riboswitches and antisense RNAs. We also highlight recent advances in RNA-based technology. The review shows that studies on both natural and synthetic systems are reciprocally beneficial.

Super-enhancer-mediated RNA processing revealed by integrative microRNA network analysis

PubMed Central

Suzuki, Hiroshi I.; Young, Richard A; Sharp, Phillip A

2017-01-01

Summary Super-enhancers are an emerging sub-class of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and a unrecognized higher-order property of super-enhancers in RNA processing beyond transcription. PMID:28283057

Non-target Effects of Green Fluorescent Protein (GFP)-derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays

PubMed Central

Nunes, Francis M. F.; Aleixo, Aline C.; Barchuk, Angel R.; Bomtorin, Ana D.; Grozinger, Christina M.; Simões, Zilá L. P.

2013-01-01

RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control. PMID:26466797

Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

PubMed

Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

2013-01-04

RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

Small Interfering RNA-Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells.

PubMed

Park, Jong-Beom; Park, Chanjoo

2017-10-01

In vitro cell culture model. To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear. Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls. Serum deprivation increased apoptosis by 40.3% ( p <0.001), decreased proliferation by 45.3% ( p <0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures ( p <0.001). Finally, Fas siRNA-mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures ( p <0.05 for both). The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

Effects of dietary octacosanol supplementation on laying performance, egg quality, serum hormone levels, and expression of genes related to the reproductive axis in laying hens.

PubMed

Long, L; Wu, S G; Yuan, F; Zhang, H J; Wang, J; Qi, G H

2017-04-01

This experiment was conducted to evaluate the effects of dietary octacosanol supplementation on laying performance, egg quality, serum hormone levels, and gene expression related to reproductive axis in laying hens to confirm the reproduction-promoting function of octacosanol. In total, 360 Hy-Line Brown (67-wk-old) laying hens were randomly assigned to one of three treatments with 0, 5, and 10 mg octacosanol (extracted from rice bran, purity >92%)/kg feed. The feeding trial lasted for 10 weeks. The results showed that the dietary addition of 5 and 10 mg/kg octacosanol improved feed efficiency by 4.9% and 3.4% (P < 0.01), increased the albumen height by 20.5% and 13.3% (P < 0.01), the Haugh unit score by 12.9% and 8.7% (P < 0.01), and the eggshell strength by 39.5% and 24.5% (P < 0.01), respectively, compared with the control diet. Dietary octacosanol addition significantly affected serum triiodothyronine, estradiol, follicle-stimulating hormone levels (P < 0.05), and progesterone and luteinizing hormone level (P < 0.01). Compared with the control, dietary addition of octacosanol at 5 mg/kg promoted the follicle-stimulating hormone receptor (FSHR) mRNA expression in different-sized follicles, and significantly increased the FSHR mRNA expression of granulosa cells from the F2 and F3 follicles (P < 0.05). Dietary supplementation with both 5 and 10 mg/kg octacosanol promoted the mRNA expression of luteinizing hormone receptor and prolactin receptor in different-sized follicles, and significantly up-regulated the expression levels in F1 granulosa cells (P < 0.05). The ovarian weight was significantly increased with the dietary addition of 5 mg/kg octacosanol (P < 0.05). The numbers of small yellow follicles and large white follicles were increased with the addition of dietary 5 and 10 mg/kg octacosanol (P < 0.01). This study provides evidence that octacosanol has the capacity to improve reproductive performance, indicating that it is a potentially effective feed additive in egg production. © 2016 Poultry Science Association Inc.

Detection of a Cis eQTL Controlling BMCO1 Gene Expression Leads to the Identification of a QTG for Chicken Breast Meat Color

PubMed Central

Le Bihan-Duval, Elisabeth; Nadaf, Javad; Berri, Cécile; Pitel, Frédérique; Graulet, Benoît; Godet, Estelle; Leroux, Sophie Y.; Demeure, Olivier; Lagarrigue, Sandrine; Duby, Cécile; Cogburn, Larry A.; Beaumont, Catherine M.; Duclos, Michel J.

2011-01-01

Classical quantitative trait loci (QTL) analysis and gene expression QTL (eQTL) were combined to identify the causal gene (or QTG) underlying a highly significant QTL controlling the variation of breast meat color in a F2 cross between divergent high-growth (HG) and low-growth (LG) chicken lines. Within this meat quality QTL, BCMO1 (Accession number GenBank: AJ271386), encoding the β-carotene 15, 15′-monooxygenase, a key enzyme in the conversion of β-carotene into colorless retinal, was a good functional candidate. Analysis of the abundance of BCMO1 mRNA in breast muscle of the HG x LG F2 population allowed for the identification of a strong cis eQTL. Moreover, reevaluation of the color QTL taking BCMO1 mRNA levels as a covariate indicated that BCMO1 mRNA levels entirely explained the variations in meat color. Two fully-linked single nucleotide polymorphisms (SNP) located within the proximal promoter of BCMO1 gene were identified. Haplotype substitution resulted in a marked difference in BCMO1 promoter activity in vitro. The association study in the F2 population revealed a three-fold difference in BCMO1 expression leading to a difference of 1 standard deviation in yellow color between the homozygous birds at this haplotype. This difference in meat yellow color was fully consistent with the difference in carotenoid content (i.e. lutein and zeaxanthin) evidenced between the two alternative haplotypes. A significant association between the haplotype, the level of BCMO1 expression and the yellow color of the meat was also recovered in an unrelated commercial broiler population. The mutation could be of economic importance for poultry production by making possible a gene-assisted selection for color, a determining aspect of meat quality. Moreover, this natural genetic diversity constitutes a new model for the study of β-carotene metabolism which may act upon diverse biological processes as precursor of the vitamin A. PMID:21750696

NIR-induced spatiotemporally controlled gene silencing by upconversion nanoparticle-based siRNA nanocarrier.

PubMed

Chen, Guojun; Ma, Ben; Xie, Ruosen; Wang, Yuyuan; Dou, Kefeng; Gong, Shaoqin

2017-12-27

Spatiotemporal control over the release or activation of biomacromolecules such as siRNA remains a significant challenge. Light-controlled release has gained popularity in recent years; however, a major limitation is that most photoactivable compounds/systems respond only to UV irradiation, but not near-infrared (NIR) light that offers a deeper tissue penetration depth and better biocompatibility. This paper reports a simple NIR-to-UV upconversion nanoparticle (UCNP)-based siRNA nanocarrier for NIR-controlled gene silencing. siRNA is complexed onto a NaYF 4 :Yb/Tm/Er UCNP through an azobenzene (Azo)-cyclodextrin (CD) host-guest interaction. The UV emission generated by the NIR-activated UCNP effectively triggers the trans-to-cis photoisomerization of azobenzene, thus leading to the release of siRNA due to unmatched host-guest pairs. The UCNP-siRNA complexes are also functionalized with PEG (i.e., UCNP-(CD/Azo)-siRNA/PEG NPs), targeting ligands (i.e., EGFR-specific GE11 peptide), acid-activatable cell-penetrating peptides (i.e., TH peptide), and imaging probes (i.e., Cy5 fluorophore). The UCNP-(CD/Azo)-siRNA/PEG NPs with both GE11 and TH peptides display a high level of cellular uptake and an excellent endosomal/lysosomal escape capability. More importantly, NIR-controlled spatiotemporal knockdown of GFP expression is successfully achieved in both a 2D monolayer cell model and a 3D multicellular tumor spheroid model. Thus, this simple and versatile nanoplatform has great potential for the selective activation or release of various biomacromolecules. Copyright © 2017. Published by Elsevier B.V.

[Methods, challenges and opportunities for big data analyses of microbiome].

PubMed

Sheng, Hua-Fang; Zhou, Hong-Wei

2015-07-01

Microbiome is a novel research field related with a variety of chronic inflamatory diseases. Technically, there are two major approaches to analysis of microbiome: metataxonome by sequencing the 16S rRNA variable tags, and metagenome by shot-gun sequencing of the total microbial (mainly bacterial) genome mixture. The 16S rRNA sequencing analyses pipeline includes sequence quality control, diversity analyses, taxonomy and statistics; metagenome analyses further includes gene annotation and functional analyses. With the development of the sequencing techniques, the cost of sequencing will decrease, and big data analyses will become the central task. Data standardization, accumulation, modeling and disease prediction are crucial for future exploit of these data. Meanwhile, the information property in these data, and the functional verification with culture-dependent and culture-independent experiments remain the focus in future research. Studies of human microbiome will bring a better understanding of the relations between the human body and the microbiome, especially in the context of disease diagnosis and therapy, which promise rich research opportunities.

A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts.

PubMed

Hoover, Malachia; Adamian, Yvess; Brown, Mark; Maawy, Ali; Chang, Alexander; Lee, Jacqueline; Gharibi, Armen; Katz, Matthew H; Fleming, Jason; Hoffman, Robert M; Bouvet, Michael; Doebler, Robert; Kelber, Jonathan A

2017-01-24

Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies.

M1 RNA is important for the in-cell solubility of its cognate C5 protein: Implications for RNA-mediated protein folding

PubMed Central

Son, Ahyun; Choi, Seong Il; Han, Gyoonhee; Seong, Baik L

2015-01-01

It is one of the fundamental questions in biology how proteins efficiently fold into their native conformations despite off-pathway events such as misfolding and aggregation in living cells. Although molecular chaperones have been known to assist the de novo folding of certain types of proteins, the role of a binding partner (or a ligand) in the folding and in-cell solubility of its interacting protein still remains poorly defined. RNase P is responsible for the maturation of tRNAs as adaptor molecules of amino acids in ribosomal protein synthesis. The RNase P from Escherichia coli, composed of M1 RNA and C5 protein, is a prototypical ribozyme in which the RNA subunit contains the catalytic activity. Using E. coli RNase P, we demonstrate that M1 RNA plays a pivotal role in the in-cell solubility of C5 protein both in vitro and in vivo. Mutations in either the C5 protein or M1 RNA that affect their interactions significantly abolished the folding of C5 protein. Moreover, we find that M1 RNA provides quality insurance of interacting C5 protein, either by promoting the degradation of C5 mutants in the presence of functional proteolytic machinery, or by abolishing their solubility if the machinery is non-functional. Our results describe a crucial role of M1 RNA in the folding, in-cell solubility, and, consequently, the proteostasis of the client C5 protein, giving new insight into the biological role of RNAs as chaperones and mediators that ensure the quality of interacting proteins. PMID:26517763

Role of Increased n-acetylaspartate Levels in Cancer

PubMed Central

Zand, Behrouz; Previs, Rebecca A.; Zacharias, Niki M.; Rupaimoole, Rajesha; Mitamura, Takashi; Nagaraja, Archana Sidalaghatta; Guindani, Michele; Dalton, Heather J.; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Hu, Wei; Pecot, Chad V.; Ivan, Cristina; Wu, Sherry Y.; McCullough, Christopher R.; Gharpure, Kshipra M.; Shoshan, Einav; Pradeep, Sunila; Mangala, Lingegowda S.; Rodriguez-Aguayo, Cristian; Wang, Ying; Nick, Alpa M.; Davies, Michael A.; Armaiz-Pena, Guillermo; Liu, Jinsong; Lutgendorf, Susan K.; Baggerly, Keith A.; Eli, Menashe Bar; Lopez-Berestein, Gabriel; Nagrath, Deepak; Bhattacharya, Pratip K.

2016-01-01

Background: The clinical and biological effects of metabolic alterations in cancer are not fully understood. Methods: In high-grade serous ovarian cancer (HGSOC) samples (n = 101), over 170 metabolites were profiled and compared with normal ovarian tissues (n = 15). To determine NAT8L gene expression across different cancer types, we analyzed the RNA expression of cancer types using RNASeqV2 data available from the open access The Cancer Genome Atlas (TCGA) website (http://www.cbioportal.org/public-portal/). Using NAT8L siRNA, molecular techniques and histological analysis, we determined cancer cell viability, proliferation, apoptosis, and tumor growth in in vitro and in vivo (n = 6–10 mice/group) settings. Data were analyzed with the Student’s t test and Kaplan-Meier analysis. Statistical tests were two-sided. Results: Patients with high levels of tumoral NAA and its biosynthetic enzyme, aspartate N-acetyltransferase (NAT8L), had worse overall survival than patients with low levels of NAA and NAT8L. The overall survival duration of patients with higher-than-median NAA levels (3.6 years) was lower than that of patients with lower-than-median NAA levels (5.1 years, P = .03). High NAT8L gene expression in other cancers (melanoma, renal cell, breast, colon, and uterine cancers) was associated with worse overall survival. NAT8L silencing reduced cancer cell viability (HEYA8: control siRNA 90.61%±2.53, NAT8L siRNA 39.43%±3.00, P < .001; A2780: control siRNA 90.59%±2.53, NAT8L siRNA 7.44%±1.71, P < .001) and proliferation (HEYA8: control siRNA 74.83%±0.92, NAT8L siRNA 55.70%±1.54, P < .001; A2780: control siRNA 50.17%±4.13, NAT8L siRNA 26.52%±3.70, P < .001), which was rescued by addition of NAA. In orthotopic mouse models (ovarian cancer and melanoma), NAT8L silencing reduced tumor growth statistically significantly (A2780: control siRNA 0.52 g±0.15, NAT8L siRNA 0.08 g±0.17, P < .001; HEYA8: control siRNA 0.79 g±0.42, NAT8L siRNA 0.24 g±0.18, P = .008, A375-SM: control siRNA 0.55 g±0.22, NAT8L siRNA 0.21 g±0.17g, P = .001). NAT8L silencing downregulated the anti-apoptotic pathway, which was mediated through FOXM1. Conclusion: These findings indicate that the NAA pathway has a prominent role in promoting tumor growth and represents a valuable target for anticancer therapy. Altered energy metabolism is a hallmark of cancer (1). Proliferating cancer cells have much greater metabolic requirements than nonproliferating differentiated cells (2,3). Moreover, altered cancer metabolism elevates unique metabolic intermediates, which can promote cancer survival and progression (4,5). Furthermore, emerging evidence suggests that proliferating cancer cells exploit alternative metabolic pathways to meet their high demand for energy and to accumulate biomass (6–8). PMID:26819345

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

PubMed

Abdel-Hadi, Ahmed M; Caley, Daniel P; Carter, David R F; Magan, Naresh

2011-06-01

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Homozygous SALL1 Mutation Causes a Novel Multiple Congenital Anomaly—Mental Retardation Syndrome

PubMed Central

Vodopiutz, Julia; Zoller, Heinz; Fenwick, Aimée L.; Arnhold, Richard; Schmid, Max; Prayer, Daniela; Müller, Thomas; Repa, Andreas; Pollak, Arnold; Aufricht, Christoph; Wilkie, Andrew O.M.; Janecke, Andreas R.

2013-01-01

Objective To delineate a novel autosomal recessive multiple congenital anomaly-mental retardation (MCA-MR) syndrome in 2 female siblings of a consanguineous pedigree and to identify the disease-causing mutation. Study design Both siblings were clinically characterized and homozygosity mapping and sequencing of candidate genes were applied. The contribution of nonsense-mediated messenger RNA (mRNA) decay to the expression of mutant mRNA in fibroblasts of a healthy carrier and a control was studied by pyrosequencing. Results We identified the first homozygous SALL1 mutation, c.3160C > T (p.R1054*), in 2 female siblings presenting with multiple congenital anomalies, central nervous system defects, cortical blindness, and absence of psychomotor development (ie, a novel recognizable, autosomal recessive MCA-MR). The mutant SALL1 transcript partially undergoes nonsense-mediated mRNA decay and is present at 43% of the normal transcript level in the fibroblasts of a healthy carrier. Conclusion Previously heterozygous SALL1 mutations and deletions have been associated with dominantly inherited anal-renal-radial-ear developmental anomalies. We identified an allelic recessive SALL1-related MCA-MR. Our findings imply that quantity and quality of SALL1 transcript are important for SALL1 function and determine phenotype, and mode of inheritance, of allelic SALL1-related disorders. This novel MCA-MR emphasizes SALL1 function as critical for normal central nervous system development and warrants a detailed neurologic investigation in all individuals with SALL1 mutations. PMID:23069192

Nucleocytoplasmic Transport of RNAs and RNA-Protein Complexes.

PubMed

Sloan, Katherine E; Gleizes, Pierre-Emmanuel; Bohnsack, Markus T

2016-05-22

RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalization ensures that sequential steps in gene expression and RNP production are performed in the correct order and it allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes, which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through nuclear pore complexes. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression under stress conditions and in disease. Copyright © 2015. Published by Elsevier Ltd.

[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].

PubMed

Hang, Xiao-Tong; Li, Jian-Dong; Zhang, Quan-Fu; Li, Chuan; Zhang, Shuo; Liang, Mi-Fang; Li, De-Xin

2010-02-01

Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.

Internal control regions for transcription of eukaryotic tRNA genes.

PubMed Central

Sharp, S; DeFranco, D; Dingermann, T; Farrell, P; Söll, D

1981-01-01

We have identified the region within a eukaryotic tRNA gene required for initiation of transcription. These results were obtained by systematically constructing deletions extending from the 5' or the 3' flanking regions into a cloned Drosophila tRNAArg gene by using nuclease BAL 31. The ability of the newly generated deletion clones to direct the in vitro synthesis of tRNA precursors was measured in transcription systems from Xenopus laevis oocytes, Drosophila Kc cells, and HeLa cells. Two control regions within the coding sequence were identified. The first was essential for transcription and was contained between nucleotides 8 and 25 of the mature tRNA sequence. Genes devoid of the second control region, which was contained between nucleotides 50 and 58 of the mature tRNA sequence, could be transcribed but with reduced efficiency. Thus, the promoter regions within a tRNA gene encode the tRNA sequences of the D stem and D loop, the invariant uridine at position 8, and the semi-invariant G-T-psi-C sequence. Images PMID:6947245

Histone Methylation and Epigenetic Silencing in Breast Cancer

DTIC Science & Technology

2010-07-01

bars show relative luciferase expression levels (firefly versus Renilla control) in SKBR3 cells treated with a control non-targeted dsRNA (NT2) and red...luciferase expression levels (firefly versus Renilla control) in SKBR3 cells treated with a control non-targeted dsRNA (NT2) and red bars depict

Plasma LncRNA-ATB, a Potential Biomarker for Diagnosis of Patients with Coal Workers' Pneumoconiosis: A Case-Control Study.

PubMed

Ma, Jixuan; Cui, Xiuqing; Rong, Yi; Zhou, Yun; Guo, Yanjun; Zhou, Min; Xiao, Lili; Chen, Weihong

2016-08-22

LncRNA-ATB (lncRNA was activated by transforming growth factor-β) has been reported to be involved in specific physiological and pathological processes in human diseases, and could serve as biomarkers for cancers. However, the role of lncRNA-ATB in coal workers' pneumoconiosis (CWP) is still unknown. This study aimed to investigate the association between lncRNA-ATB and CWP. Quantitative real-time polymerase chain reaction was performed to detect plasma lncRNA-ATB expression in 137 CWP patients, 72 healthy coal miners and 168 healthy controls. LncRNA-ATB was significantly upregulated in CWP (p < 0.05). Compared with the healthy controls and healthy coal miners, the odds ratios (ORs) (95% confidence interval (CI)) for CWP were 2.57 (1.52-4.33) and 2.17 (1.04-4.53), respectively. LncRNA-ATB was positively associated with transforming growth factor-β1 (TGF-β1) (r = 0.30, p = 0.003) and negative correlated with vital capacity (VC) (r = -0.18, p = 0.033) and forced vital capacity (FVC) (r = -0.18, p = 0.046) in CWP patients. Compared with healthy controls, the area under the curve (AUC) was 0.84, resulting in a 71.17% sensitivity and 88.14% specificity. When compared with healthy coal miners, the AUC was 0.83, the sensitivity and specificity were 70.07% and 86.36%, respectively. LncRNA-ATB expression is commonly increased in CWP and significantly correlates with the TGF-β1 in CWP patients. Furthermore, elevated lncRNA-ATB was associated with CWP risk and may serve as a potential biomarker for CWP.

RNA-seq for gene identification and transcript profiling in relation to root growth of bermudagrass (Cynodon dactylon) under salinity stress.

PubMed

Hu, Longxing; Li, Huiying; Chen, Liang; Lou, Yanhong; Amombo, Erick; Fu, Jinmin

2015-08-04

Soil salinity is one of the most significant abiotic stresses affecting plant shoots and roots growth. The adjustment of root architecture to spatio-temporal heterogeneity in salinity is particularly critical for plant growth and survival. Bermudagrass (Cynodon dactylon) is a widely used turf and forage perennial grass with a high degree of salinity tolerance. Salinity appears to stimulate the growth of roots and decrease their mortality in tolerant bermudagrass. To estimate a broad spectrum of genes related to root elongation affected by salt stress and the molecular mechanisms that control the positive response of root architecture to salinity, we analyzed the transcriptome of bermudagrass root tips in response to salinity. RNA-sequencing was performed in root tips of two bermudagrass genotypes contrasting in salt tolerance. A total of 237,850,130 high quality clean reads were generated and 250,359 transcripts were assembled with an average length of 1115 bp. Totally, 103,324 unigenes obtained with 53,765 unigenes (52 %) successfully annotated in databases. Bioinformatics analysis indicated that major transcription factor (TF) families linked to stress responses and growth regulation (MYB, bHLH, WRKY) were differentially expressed in root tips of bermudagrass under salinity. In addition, genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases) were identified. RNA-seq analysis identified candidate genes encoding TFs involved in the regulation of lignin synthesis, reactive oxygen species (ROS) homeostasis controlled by peroxidases, and the regulation of phytohormone signaling that promote cell wall loosening and therefore root growth under salinity.

Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W.

PubMed

Yu, Tsong-Ann; Chiang, Chu-Hui; Wu, Hui-Wen; Li, Chin-Mei; Yang, Ching-Fu; Chen, Jun-Han; Chen, Yu-Wen; Yeh, Shyi-Dong

2011-03-01

Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding.

Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

PubMed

Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

2015-07-01

The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs. Copyright © 2015 Elsevier B.V. All rights reserved.

EOL-1, the homolog of the mammalian Dom3Z, regulates olfactory learning in C. elegans.

PubMed

Shen, Yu; Zhang, Jiangwen; Calarco, John A; Zhang, Yun

2014-10-01

Learning is an essential function of the nervous system. However, our understanding of molecular underpinnings of learning remains incomplete. Here, we characterize a conserved protein EOL-1 that regulates olfactory learning in Caenorhabditis elegans. A recessive allele of eol-1 (enhanced olfactory learning) learns better to adjust its olfactory preference for bacteria foods and eol-1 acts in the URX sensory neurons to regulate learning. The mammalian homolog of EOL-1, Dom3Z, which regulates quality control of pre-mRNAs, can substitute the function of EOL-1 in learning regulation, demonstrating functional conservation between these homologs. Mutating the residues of Dom3Z that are critical for its enzymatic activity, and the equivalent residues in EOL-1, abolishes the function of these proteins in learning. Together, our results provide insights into the function of EOL-1/Dom3Z and suggest that its activity in pre-mRNA quality control is involved in neural plasticity. Copyright © 2014 the authors 0270-6474/14/3413364-07$15.00/0.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

PubMed

Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

2017-10-01

Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

The centrality of RNA for engineering gene expression

PubMed Central

Chappell, James; Takahashi, Melissa K; Meyer, Sarai; Loughrey, David; Watters, Kyle E; Lucks, Julius

2013-01-01

Synthetic biology holds promise as both a framework for rationally engineering biological systems and a way to revolutionize how we fundamentally understand them. Essential to realizing this promise is the development of strategies and tools to reliably and predictably control and characterize sophisticated patterns of gene expression. Here we review the role that RNA can play towards this goal and make a case for why this versatile, designable, and increasingly characterizable molecule is one of the most powerful substrates for engineering gene expression at our disposal. We discuss current natural and synthetic RNA regulators of gene expression acting at key points of control – transcription, mRNA degradation, and translation. We also consider RNA structural probing and computational RNA structure predication tools as a way to study RNA structure and ultimately function. Finally, we discuss how next-generation sequencing methods are being applied to the study of RNA and to the characterization of RNA's many properties throughout the cell. PMID:24124015

Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR.

PubMed

Du, Yishuai; Zhang, Linlin; Xu, Fei; Huang, Baoyu; Zhang, Guofan; Li, Li

2013-03-01

Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1α (EF-1α), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in OsHV-1 infected larvae. These high quality internal controls will be a valuable resource in future studies of oyster larval mortality. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interaction of microRNA-21/145 and Smad3 domain-specific phosphorylation in hepatocellular carcinoma

PubMed Central

Wang, Ji Yu; Fang, Meng; Boye, Alex; Wu, Chao; Wu, Jia Jun; Ma, Ying; Hou, Shu; Kan, Yue; Yang, Yan

2017-01-01

MicroRNAs 21 and 145 exhibit inverse expression in Hepatocellular carcinoma (HCC), but how they relate to Smad3 C-terminal and Link region phosphorylation (pSmad3C and pSmad3L) downstream of TGF-β/MAPK signaling, remains inconclusive. Our results suggest microRNA-145 targets Smad3 in HepG2 cells. Decreased tumor volume and increased apoptosis were produced in both microRNA-21 antagomir and microRNA-145 agomir groups compared to controls. Inhibition of TβRI and MAPK (ERK, JNK, and p38) activation respectively produced decreased microRNA-21 but increased microRNA-145 expression. Correspondingly, the expression level of pSmad3C obviously increased while pSmad3L decreased in microRNA-145 agomir-group and the expression of pSmad3C/3L were not markedly changed but pERK, pJNK, pp38 decreased in microRNA-21 antagomir-group compared to controls. On the other hand, microRNA-145 and 21 increased respectively in xenografts of HepG2 cells transfected with Smad3 EPSM and 3S-A plasmid, and this correlated with the overexpression of pSmad3C and pSmad3L respectively compared to control. To conclude, microRNA-21 promotes tumor progression in a MAPK-dependent manner while microRNA-145 suppresses it via domain-specific phosphorylation of Smad3 in HCC. Meanwhile, increased pSmad3C/3L lead to the up-regulation of microRNA-145/21 respectively. The interaction between pSmad3C/3L and microRNA-145/21 regulates HCC progression and the switch of pSmad3C/3L may serve as an important target for HCC therapy. PMID:29156696

Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation.

PubMed

Robbins, Marjorie; Judge, Adam; Ambegia, Ellen; Choi, Catherine; Yaworski, Ed; Palmer, Lorne; McClintock, Kevin; MacLachlan, Ian

2008-10-01

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

PubMed

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2018-03-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.

PubMed Central

Tabor, S; Richardson, C C

1985-01-01

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase. Images PMID:3156376

Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of influenza virus and severe acute respiratory syndrome coronavirus.

PubMed

Yu, Xin-Fen; Pan, Jing-Cao; Ye, Rong; Xiang, Hai-Qing; Kou, Yu; Huang, Zhi-Cheng

2008-03-01

The common respiratory viruses, including influenza A, influenza B, and newly emerging severe acute respiratory syndrome (SARS) viruses, may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required for single clinical samples. In addition, there is an urgent need for noninfectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR (RT-PCR) targeting of the RNAs of influenza A and influenza B viruses and SARS coronavirus was performed, and the resulting products were spliced into a fragment which was packaged into armored RNA for use as a noninfectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control and the three RNA viruses could be detected simultaneously in a single reaction mix. The detection limit of the multiplex real-time PCR was 10 copies/microl of armored RNA.

Existence of a microRNA pathway in anucleate platelets

PubMed Central

Landry, Patricia; Plante, Isabelle; Ouellet, Dominique L; Perron, Marjorie P; Rousseau, Guy; Provost, Patrick

2010-01-01

Platelets play a critical role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion that underlie stroke and acute coronary syndromes. Anucleate platelets contain messenger RNAs (mRNAs) and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation. Further analyses revealed that platelets contain Dicer and Argonaute 2 (Ago2) complexes functional in exogenously supplied miRNA precursor (pre-miRNA) processing and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y12 mRNA in Ago2 immunoprecipitates suggests that P2Y12 expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. PMID:19668211

Morphometric and molecular data on two mitochondrial genes of a newly discovered chimaeran fish ( Hydrolagus melanophasma, Chondrichthyes)

NASA Astrophysics Data System (ADS)

De La Cruz-Agüero, José; García-Rodríguez, Francisco Javier; Cota-Gómez, Víctor Manuel; Melo-Barrera, Felipe Neri; González-Armas, Rogelio

2012-06-01

Fresh and preserved (type material) specimens of the black ghost chimaera Hydrolagus melanophasma were compared for morphometric characteristics. A molecular comparison was also performed on two mitochondrial gene sequences (12S rRNA and 16S rRNA gene sequences). While significant differences in measurements were found, the differences were not attributable to sexual dimorphism or the quality of the specimens, but to the sample size and the type of statistical tests. The result of the genetic characterization showed that 12S rRNA and 16S rRNA genes represented robust molecular markers that characterized the species.

Determination of the conformational ensemble of the TAR RNA by X-ray scattering interferometry.

PubMed

Shi, Xuesong; Walker, Peter; Harbury, Pehr B; Herschlag, Daniel

2017-05-05

The conformational ensembles of structured RNA's are crucial for biological function, but they remain difficult to elucidate experimentally. We demonstrate with HIV-1 TAR RNA that X-ray scattering interferometry (XSI) can be used to determine RNA conformational ensembles. X-ray scattering interferometry (XSI) is based on site-specifically labeling RNA with pairs of heavy atom probes, and precisely measuring the distribution of inter-probe distances that arise from a heterogeneous mixture of RNA solution structures. We show that the XSI-based model of the TAR RNA ensemble closely resembles an independent model derived from NMR-RDC data. Further, we show how the TAR RNA ensemble changes shape at different salt concentrations. Finally, we demonstrate that a single hybrid model of the TAR RNA ensemble simultaneously fits both the XSI and NMR-RDC data set and show that XSI can be combined with NMR-RDC to further improve the quality of the determined ensemble. The results suggest that XSI-RNA will be a powerful approach for characterizing the solution conformational ensembles of RNAs and RNA-protein complexes under diverse solution conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

RNA transcriptional biosignature analysis for identifying febrile infants with serious bacterial infections in the emergency department: a feasibility study.

PubMed

Mahajan, Prashant; Kuppermann, Nathan; Suarez, Nicolas; Mejias, Asuncion; Casper, Charlie; Dean, J Michael; Ramilo, Octavio

2015-01-01

To develop the infrastructure and demonstrate the feasibility of conducting microarray-based RNA transcriptional profile analyses for the diagnosis of serious bacterial infections in febrile infants 60 days and younger in a multicenter pediatric emergency research network. We designed a prospective multicenter cohort study with the aim of enrolling more than 4000 febrile infants 60 days and younger. To ensure success of conducting complex genomic studies in emergency department (ED) settings, we established an infrastructure within the Pediatric Emergency Care Applied Research Network, including 21 sites, to evaluate RNA transcriptional profiles in young febrile infants. We developed a comprehensive manual of operations and trained site investigators to obtain and process blood samples for RNA extraction and genomic analyses. We created standard operating procedures for blood sample collection, processing, storage, shipping, and analyses. We planned to prospectively identify, enroll, and collect 1 mL blood samples for genomic analyses from eligible patients to identify logistical issues with study procedures. Finally, we planned to batch blood samples and determined RNA quantity and quality at the central microarray laboratory and organized data analysis with the Pediatric Emergency Care Applied Research Network data coordinating center. Below we report on establishment of the infrastructure and the feasibility success in the first year based on the enrollment of a limited number of patients. We successfully established the infrastructure at 21 EDs. Over the first 5 months we enrolled 79% (74 of 94) of eligible febrile infants. We were able to obtain and ship 1 mL of blood from 74% (55 of 74) of enrolled participants, with at least 1 sample per participating ED. The 55 samples were shipped and evaluated at the microarray laboratory, and 95% (52 of 55) of blood samples were of adequate quality and contained sufficient RNA for expression analysis. It is possible to create a robust infrastructure to conduct genomic studies in young febrile infants in the context of a multicenter pediatric ED research setting. The sufficient quantity and high quality of RNA obtained suggests that whole blood transcriptional profile analysis for the diagnostic evaluation of young febrile infants can be successfully performed in this setting.

Negative regulators of the RIG-I-like receptor signaling pathway

PubMed Central

Quicke, Kendra M.; Diamond, Michael S.; Suthar, Mehul S.

2017-01-01

SUMMARY Upon recognition of specific molecular patterns on viruses, bacteria and fungi, host cells trigger an innate immune response, which culminates in the production of type I interferons (IFN), pro-inflammatory cytokines and chemokines, and restricts pathogen replication and spread within the host. At each stage of the immune response, there are stimulatory and inhibitory signals that regulate the magnitude, quality, and character of the response. Positive regulation promotes an antiviral state to control and eventually clear infection whereas negative regulation dampens inflammation and prevents immune-mediated tissue damage. An over-exuberant innate immune response can lead to the destruction of cells and tissues, and the development of spontaneous autoimmunity. The RIG-I-like receptors (RLRs) retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) belong to a family of cytosolic host RNA helicases that recognize distinct non-self RNA signatures and trigger innate immune responses against several RNA virus infections. The RLR signaling pathway is tightly regulated to achieve a well-orchestrated response aimed at maximizing antiviral immunity and minimizing immune-mediated pathology. This review highlights contemporary findings on negative regulators of the RLR signaling pathway, with specific focus on the proteins and biological processes that directly regulate RIG-I, MDA5 and MAVS function. PMID:28295214

Modification of Monolignol Biosynthetic Pathway in Jute: Different Gene, Different Consequence

PubMed Central

Shafrin, Farhana; Ferdous, Ahlan Sabah; Sarkar, Suprovath Kumar; Ahmed, Rajib; Amin, Al-; Hossain, Kawsar; Sarker, Mrinmoy; Rencoret, Jorge; Gutiérrez, Ana; del Rio, Jose C.; Sanan-Mishra, Neeti; Khan, Haseena

2017-01-01

Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16–25% reduction in acid insoluble lignin for the whole stem and ~13–14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition. PMID:28051165

Fidgetin-like 2: a microtubule-based regulator of wound healing

PubMed Central

Charafeddine, Rabab A.; Makdisi, Joy; Schairer, David; O’Rourke, Brian P.; Diaz-Valencia, Juan D.; Chouake, Jason; Kutner, Allison; Krausz, Aimee; Adler, Brandon; Nacharaju, Parimala; Liang, Hongying; Mukherjee, Suranjana; Friedman, Joel M.; Friedman, Adam; Nosanchuk, Joshua D.; Sharp, David J.

2015-01-01

Wound healing is a complex process driven largely by the migration of a variety of distinct cell types from the wound margin into the wound zone. In this study, we identify the previously uncharacterized microtubule-severing enzyme, Fidgetin-like 2 (FL2), as a fundamental regulator of cell migration that can be targeted in vivo using nanoparticle-encapsulated siRNA to promote wound closure and regeneration. In vitro, depletion of FL2 from mammalian tissue culture cells results in a more than two-fold increase in the rate of cell movement, due in part to a significant increase in directional motility. Immunofluorescence analyses indicate that FL2 normally localizes to the cell edge, importantly to the leading edge of polarized cells, where it regulates the organization and dynamics of the microtubule cytoskeleton. To clinically translate these findings, we utilized a nanoparticle-based siRNA delivery platform to locally deplete FL2 in both murine full-thickness excisional and burn wounds. Topical application of FL2 siRNA nanoparticles to either wound type results in a significant enhancement in the rate and quality of wound closure both clinically and histologically relative to controls. Taken together, these results identify FL2 as a promising therapeutic target to promote the regeneration and repair of cutaneous wounds. PMID:25756798

Autoregulatory mechanisms controlling the Microprocessor.

PubMed

Triboulet, Robinson; Gregory, Richard I

2010-01-01

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature 22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part ofa newly identified regulatory mechanism controlling Microprocessor activity.

Complete mitochondrial genome of the desert toad-headed agama, Phrynocephalus przewalskii (Reptilia, Squamata, Agamidae), a novel gene organization in vertebrate mtDNA.

PubMed

Li, Donghai; Song, Sen; Chen, Tao; Zhang, Caihong; Chang, Cheng

2015-01-01

The mitogenome of the desert toad-headed agama, Phrynocephalus przewalskii, was amplified using polymerase chain reaction (PCR), long-and-accurate PCR and directly sequenced by primer walking. The complete mitogenome was 16,892 bp in size and contained 13 protein-coding, 23 tRNA, and 2 rRNA genes, and 1 control region. The mitogenome of the P. przewalskii was similar to those of other Phrynocephalus sand lizards in gene arrangement and composition, except that tRNA-Phe and tRNA-Pro were exchanged and tRNA-Phe had two copies. The control region comprised three parts, one between tRNA-Thr and tRNA-Phe, a second between the two tRNA-Phe copies, and a third between tRNA-Pro and 12S RNA. The overall nucleotide composition of the H-strand was 36.3% A, 26.7% T, 12.5% G, 24.6% C. The complete mitogenome of P. przewalskii will contribute to understanding the evolution of the genus Phrynocephalus and the family Agamidae.

The Use of EST Expression Matrixes for the Quality Control of Gene Expression Data

PubMed Central

Milnthorpe, Andrew T.; Soloviev, Mikhail

2012-01-01

EST expression profiling provides an attractive tool for studying differential gene expression, but cDNA libraries' origins and EST data quality are not always known or reported. Libraries may originate from pooled or mixed tissues; EST clustering, EST counts, library annotations and analysis algorithms may contain errors. Traditional data analysis methods, including research into tissue-specific gene expression, assume EST counts to be correct and libraries to be correctly annotated, which is not always the case. Therefore, a method capable of assessing the quality of expression data based on that data alone would be invaluable for assessing the quality of EST data and determining their suitability for mRNA expression analysis. Here we report an approach to the selection of a small generic subset of 244 UniGene clusters suitable for identification of the tissue of origin for EST libraries and quality control of the expression data using EST expression information alone. We created a small expression matrix of UniGene IDs using two rounds of selection followed by two rounds of optimisation. Our selection procedures differ from traditional approaches to finding “tissue-specific” genes and our matrix yields consistency high positive correlation values for libraries with confirmed tissues of origin and can be applied for tissue typing and quality control of libraries as small as just a few hundred total ESTs. Furthermore, we can pick up tissue correlations between related tissues e.g. brain and peripheral nervous tissue, heart and muscle tissues and identify tissue origins for a few libraries of uncharacterised tissue identity. It was possible to confirm tissue identity for some libraries which have been derived from cancer tissues or have been normalised. Tissue matching is affected strongly by cancer progression or library normalisation and our approach may potentially be applied for elucidating the stage of normalisation in normalised libraries or for cancer staging. PMID:22412959

Genome-Wide Identification of miRNAs Responsive to Drought in Peach (Prunus persica) by High-Throughput Deep Sequencing

PubMed Central

Eldem, Vahap; Çelikkol Akçay, Ufuk; Ozhuner, Esma; Bakır, Yakup; Uranbey, Serkan; Unver, Turgay

2012-01-01

Peach (Prunus persica L.) is one of the most important worldwide fresh fruits. Since fruit growth largely depends on adequate water supply, drought stress is considered as the most important abiotic stress limiting fleshy fruit production and quality in peach. Plant responses to drought stress are regulated both at transcriptional and post-transcriptional level. As post-transcriptional gene regulators, miRNAs (miRNAs) are small (19–25 nucleotides in length), endogenous, non-coding RNAs. Recent studies indicate that miRNAs are involved in plant responses to drought. Therefore, Illumina deep sequencing technology was used for genome-wide identification of miRNAs and their expression profile in response to drought in peach. In this study, four sRNA libraries were constructed from leaf control (LC), leaf stress (LS), root control (RC) and root stress (RS) samples. We identified a total of 531, 471, 535 and 487 known mature miRNAs in LC, LS, RC and RS libraries, respectively. The expression level of 262 (104 up-regulated, 158 down-regulated) of the 453 miRNAs changed significantly in leaf tissue, whereas 368 (221 up-regulated, 147 down-regulated) of the 465 miRNAs had expression levels that changed significantly in root tissue upon drought stress. Additionally, a total of 197, 221, 238 and 265 novel miRNA precursor candidates were identified from LC, LS, RC and RS libraries, respectively. Target transcripts (137 for LC, 133 for LS, 148 for RC and 153 for RS) generated significant Gene Ontology (GO) terms related to DNA binding and catalytic activites. Genome-wide miRNA expression analysis of peach by deep sequencing approach helped to expand our understanding of miRNA function in response to drought stress in peach and Rosaceae. A set of differentially expressed miRNAs could pave the way for developing new strategies to alleviate the adverse effects of drought stress on plant growth and development. PMID:23227166

La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region

PubMed Central

Philippe, Lucas; Vasseur, Jean-Jacques; Debart, Françoise

2018-01-01

Abstract Cell growth is a complex process shaped by extensive and coordinated changes in gene expression. Among these is the tightly regulated translation of a family of growth-related mRNAs defined by a 5′ terminal oligopyrimidine (TOP) motif. TOP mRNA translation is partly controlled via the eukaryotic initiation factor 4F (eIF4F), a translation factor that recognizes the mRNA 5′ cap structure. Recent studies have also implicated La-related protein 1 (LARP1), which competes with eIF4F for binding to mRNA 5′ ends. However, it has remained controversial whether LARP1 represses TOP mRNA translation directly and, if so, what features define its mRNA targets. Here, we show that the C-terminal half of LARP1 is necessary and sufficient to control TOP mRNA translation in cells. This fragment contains the DM15 cap-binding domain as well as an adjacent regulatory region that we identified. We further demonstrate that purified LARP1 represses TOP mRNA translation in vitro through the combined recognition of both the TOP sequence and cap structure, and that its intrinsic repressive activity and affinity for these features are subject to regulation. These results support a model whereby the translation of TOP mRNAs is controlled by a growth-regulated competition between eIF4F and LARP1 for their 5′ ends. PMID:29244122

Narcolepsy patients' blood-based miRNA expression profiling: miRNA expression differences with Pandemrix vaccination.

PubMed

Mosakhani, N; Sarhadi, V; Panula, P; Partinen, M; Knuutila, S

2017-11-01

Narcolepsy is a neurological sleep disorder characterized by excessive daytime sleepiness and nighttime sleep disturbance. Among children and adolescents vaccinated with Pandemrix vaccine in Finland and Sweden, the number of narcolepsy cases increased. Our aim was to identify miRNAs involved in narcolepsy and their association with Pandemrix vaccination. We performed global miRNA proofing by miRNA microarrays followed by RT-PCR verification on 20 narcolepsy patients (Pandemrix-associated and Pandemrix-non-associated) and 17 controls (vaccinated and non-vaccinated). Between all narcolepsy patients and controls, 11 miRNAs were differentially expressed; 17 miRNAs showed significantly differential expression between Pandemrix-non-associated narcolepsy patients and non-vaccinated healthy controls. MiR-188-5p and miR-4499 were over-expressed in narcolepsy patients vs healthy controls. Two miRNAs, miR-1470 and miR-4455, were under-expressed in Pandemrix-associated narcolepsy patients vs Pandemrix-non-associated narcolepsy patients. We identified miRNA expression patterns in narcolepsy patients that linked them to mRNA targets known to be involved in brain-related pathways or brain disorders. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Developmental transcriptome profiling of bovine muscle tissue reveals an abundant GosB that regulates myoblast proliferation and apoptosis

PubMed Central

Yang, Jiameng; Dong, Dong; Huang, Yongzhen; Lan, Xianyong; Plath, Martin; Lei, Chuzhao; Qi, Xinglei; Bai, Yueyu; Chen, Hong

2017-01-01

The formation of bovine skeletal muscle involves complex developmental and physiological processes that play a vital role in determining the quality of beef; however, the regulatory mechanisms underlying differences in meat quality are largely unknown. We conducted transcriptome analysis of bovine muscle tissues to compare gene expression profiles between embryonic and adult stages. Total RNAs from skeletal muscle of Qinchuan cattle at fetal and adult stages were used to construct libraries for Illumina next-generation sequencing using the Ribo-Zero RNA sequencing (RNA-Seq) method. We found a total of 19,695 genes to be expressed in fetal and adult stages, whereby 3,299 were expressed only in fetal, and 433 only in adult tissues. We characterized the role of a candidate gene (GosB), which was highly (but differentially) expressed in embryonic and adult skeletal muscle tissue. GosB increased the number of myoblasts in the S-phase of the cell cycle, and decreased the proportion of cells in the G0/G1 phase. GosB promoted the proliferation of myoblasts and protected them from apoptosis via regulating Bcl-2 expression and controlling the intracellular calcium concentration. Modulation of GosB expression in muscle tissue may emerge as a potential target in breeding strategies attempting to alter myoblast numbers in cattle. PMID:28404879

MD-miniRNA could be a more accurate biomarker for prostate cancer screening compared with serum prostate-specific antigen level.

PubMed

Xue, Dong; Zhou, Cui-Xing; Shi, Yun-Bo; Lu, Hao; He, Xiao-Zhou

2015-05-01

Prostate cancer and prostatic hyperplasia detection remains a great challenge, lacking of effective non-invasive and specific diagnostic biomarkers. In the current study, we aimed to identify the relative expression of plasma MD-miniRNA and its diagnostic performance in differentiating prostate cancer and prostatic hyperplasia patients from healthy controls, compared with serum prostate-specific antigen (PSA) level. All of the clinical participants (63 prostate cancer patients, 32 prostatic hyperplasia patients, and 50 healthy controls) were obtained from the Third Affiliated Hospital of Suzhou University in China between January 2013 and April 2014. Clinical characteristics were well matched. Plasma samples were extracted to test the relative expression of MD-miniRNA using the method of qRT-PCR. SPSS 22.0 statistical software package was used to analyze the data and GraphPad Prism 6.0 was used to generate the graphs. Relativity expression of plasma MD-miniRNA was significantly upregulated in prostate cancer, compared with prostatic hyperplasia patients and healthy controls. Serum PSA level revealed similar differences among these groups. MD-miniRNA presented a relatively high diagnostic accuracy with AUC of 0.86 (95 % CI 0.80-0.93) in differentiating prostate cancer patients from healthy controls. Simultaneously, MD-miniRNA was able to discriminate prostate cancer patients from prostatic hyperplasia controls with AUC of 0.79 (95 % CI 0.70-0.88). In addition, MD-miniRNA displayed a better diagnostic performance than PSA level. However, the panel of these two biomarkers revealed the best diagnostic performance, compared with either single biomarker. Results of this study showed that plasma MD-miniRNA could serve as a promising and noninvasive biomarker for diagnosing prostate cancer. Further large-scale studies are needed to confirm its clinical diagnosis accuracy.

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.