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  1. Article ; Online: Microarray Analysis of Whole-Transcriptome RNAs Including Non-Coding RNAs.

    Thuillier, Quentin / Behm-Ansmant, Isabelle

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2300, Page(s) 143–164

    Abstract: Non-coding RNAs participate in most cellular processes and play a causative role in several diseases. In addition to their relevance as targets or tools for therapy, ncRNAs have been extensively detected in body fluids supporting their role as easily ... ...

    Abstract Non-coding RNAs participate in most cellular processes and play a causative role in several diseases. In addition to their relevance as targets or tools for therapy, ncRNAs have been extensively detected in body fluids supporting their role as easily accessible and minimally invasive biomarkers. However, the precise measurement of circulating ncRNAs remains challenging due to their low abundance and the heterogeneity of the ncRNA population (size, polyadenylation status, circular forms). Microarrays constitute a very powerful method to analyze the expression level and the splicing pattern of circulating ncRNAs since they preserve sample integrity (no need to remove globin or rRNA) and allow precise quantification of low-abundance transcripts (no limitation by read depth). This chapter describes the protocols used in our lab to extract and purify total RNAs from PAXgene RNA Blood Tubes and to perform RNA labeling and hybridization on the Clariom™ D microarrays from Affymetrix.
    MeSH term(s) Blood Specimen Collection ; Gene Expression Profiling ; Humans ; Oligonucleotide Array Sequence Analysis/methods ; RNA, Untranslated/blood ; RNA, Untranslated/chemistry ; RNA, Untranslated/genetics ; Staining and Labeling ; Whole Exome Sequencing/methods
    Chemical Substances RNA, Untranslated
    Language English
    Publishing date 2021-04-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1386-3_14
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Study of Genome-Wide Occupancy of Long Non-Coding RNAs Using Chromatin Isolation by RNA Purification (ChIRP).

    Alfeghaly, Charbel / Behm-Ansmant, Isabelle / Maenner, Sylvain

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2300, Page(s) 107–117

    Abstract: Long noncoding RNAs (lncRNAs) have recently emerged as masters of gene expression regulation by exerting their functions in all cell compartments through a wide repertoire of mechanisms. A high portion of lncRNAs are robustly enriched in the chromatin ... ...

    Abstract Long noncoding RNAs (lncRNAs) have recently emerged as masters of gene expression regulation by exerting their functions in all cell compartments through a wide repertoire of mechanisms. A high portion of lncRNAs are robustly enriched in the chromatin fraction suggesting a broad regulatory role in the nuclear compartment. Despite the advances in this field, the interaction between lncRNAs and the chromatin is still poorly understood. This led to the emergence of numerous hybridization capture assays such as the Chromatin Isolation by RNA Purification (ChIRP) which revealed at high resolution the genomic binding sites of several nuclear lncRNAs. In this chapter, we describe the ChIRP protocol that was successfully applied to the lncRNA ANRIL. We also provide a user-friendly bioinformatic pipeline for ChIRP-seq data analysis.
    MeSH term(s) Binding Sites ; Chromatin/chemistry ; Chromatin/genetics ; Gene Expression Regulation ; Genome, Human ; HEK293 Cells ; Humans ; Nucleic Acid Hybridization/methods ; RNA, Long Noncoding/analysis ; Sequence Analysis, RNA ; Workflow
    Chemical Substances CDKN2B antisense RNA, human ; Chromatin ; RNA, Long Noncoding
    Language English
    Publishing date 2021-04-01
    Publishing country United States
    Document type Journal Article
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1386-3_11
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  3. Article ; Online: Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5' Adaptor Ligation.

    Vautrot, Valentin / Behm-Ansmant, Isabelle

    Methods in molecular biology (Clifton, N.J.)

    2019  Volume 2065, Page(s) 39–54

    Abstract: Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a ... ...

    Abstract Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan
    MeSH term(s) Animals ; Fluorescent Dyes/chemistry ; Humans ; Mice ; MicroRNAs/chemistry ; MicroRNAs/genetics ; MicroRNAs/isolation & purification ; Molecular Probe Techniques/instrumentation ; Molecular Probes/chemistry ; RNA, Messenger/chemistry ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction/instrumentation ; Real-Time Polymerase Chain Reaction/methods ; Reverse Transcription ; Sensitivity and Specificity
    Chemical Substances Fluorescent Dyes ; MicroRNAs ; Molecular Probes ; RNA, Messenger
    Language English
    Publishing date 2019-10-02
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-9833-3_4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Fluorescence In Situ Hybridization of Small Non-Coding RNAs.

    Vautrot, Valentin / Heckler, Géraud / Aigueperse, Christelle / Behm-Ansmant, Isabelle

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2300, Page(s) 73–85

    Abstract: The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules ... ...

    Abstract The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor
    MeSH term(s) Biotin/chemistry ; Fluoresceins/chemistry ; Gene Expression ; HeLa Cells ; Humans ; In Situ Hybridization, Fluorescence/methods ; RNA, Small Untranslated/analysis ; RNA, Small Untranslated/chemistry ; RNA, Small Untranslated/genetics ; Streptavidin/chemistry ; Sulfonic Acids/chemistry
    Chemical Substances Fluoresceins ; RNA, Small Untranslated ; Sulfonic Acids ; alexa fluor 488 ; Biotin (6SO6U10H04) ; Streptavidin (9013-20-1)
    Language English
    Publishing date 2021-04-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1386-3_8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection.

    Alfeghaly, Charbel / Aigueperse, Christelle / Maenner, Sylvain / Behm-Ansmant, Isabelle

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2300, Page(s) 31–37

    Abstract: The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA ... ...

    Abstract The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and Lipofectamine
    MeSH term(s) Calcium Phosphates/chemistry ; Gene Silencing ; HEK293 Cells ; HeLa Cells ; Humans ; Lipids/chemistry ; Oligonucleotides, Antisense/pharmacology ; RNA, Long Noncoding/genetics ; Transfection/methods
    Chemical Substances CDKN2B antisense RNA, human ; Calcium Phosphates ; Lipids ; Lipofectamine ; Oligonucleotides, Antisense ; RNA, Long Noncoding ; calcium phosphate (97Z1WI3NDX)
    Language English
    Publishing date 2021-04-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1386-3_4
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article: The Normal, the Radiosensitive, and the Ataxic in the Era of Precision Radiotherapy: A Narrative Review.

    Pereira, Sandrine / Orlandi, Ester / Deneuve, Sophie / Barcellini, Amelia / Chalaszczyk, Agnieszka / Behm-Ansmant, Isabelle / Hettal, Liza / Rancati, Tiziana / Vogin, Guillaume / Thariat, Juliette

    Cancers

    2022  Volume 14, Issue 24

    Abstract: 1) Background: radiotherapy is a cornerstone of cancer treatment. When delivering a tumoricidal dose, the risk of severe late toxicities is usually kept below 5% using dose-volume constraints. However, individual radiation sensitivity (iRS) is ... ...

    Abstract (1) Background: radiotherapy is a cornerstone of cancer treatment. When delivering a tumoricidal dose, the risk of severe late toxicities is usually kept below 5% using dose-volume constraints. However, individual radiation sensitivity (iRS) is responsible (with other technical factors) for unexpected toxicities after exposure to a dose that induces no toxicity in the general population. Diagnosing iRS before radiotherapy could avoid unnecessary toxicities in patients with a grossly normal phenotype. Thus, we reviewed iRS diagnostic data and their impact on decision-making processes and the RT workflow; (2) Methods: following a description of radiation toxicities, we conducted a critical review of the current state of the knowledge on individual determinants of cellular/tissue radiation; (3) Results: tremendous advances in technology now allow minimally-invasive genomic, epigenetic and functional testing and a better understanding of iRS. Ongoing large translational studies implement various tests and enriched NTCP models designed to improve the prediction of toxicities. iRS testing could better support informed radiotherapy decisions for individuals with a normal phenotype who experience unusual toxicities. Ethics of medical decisions with an accurate prediction of personalized radiotherapy's risk/benefits and its health economics impact are at stake; (4) Conclusions: iRS testing represents a critical unmet need to design personalized radiotherapy protocols relying on extended NTCP models integrating iRS.
    Language English
    Publishing date 2022-12-19
    Publishing country Switzerland
    Document type Journal Article ; Review
    ZDB-ID 2527080-1
    ISSN 2072-6694
    ISSN 2072-6694
    DOI 10.3390/cancers14246252
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  7. Article ; Online: Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images.

    Heckler, Géraud / Aigueperse, Christelle / Hettal, Liza / Thuillier, Quentin / de Chaumont, Fabrice / Dallongeville, Stéphane / Behm-Ansmant, Isabelle

    Journal of visualized experiments : JoVE

    2020  , Issue 161

    Abstract: The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the ... ...

    Abstract The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.
    MeSH term(s) Cell Body/metabolism ; Cell Nucleus ; Humans ; Microscopy, Fluorescence/methods ; Nuclear Proteins ; Workflow
    Chemical Substances Nuclear Proteins
    Language English
    Publishing date 2020-07-15
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
    ZDB-ID 2259946-0
    ISSN 1940-087X ; 1940-087X
    ISSN (online) 1940-087X
    ISSN 1940-087X
    DOI 10.3791/60990
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Radiomics Method for the Differential Diagnosis of Radionecrosis Versus Progression after Fractionated Stereotactic Body Radiotherapy for Brain Oligometastasis.

    Hettal, Liza / Stefani, Anais / Salleron, Julia / Courrech, Florent / Behm-Ansmant, Isabelle / Constans, Jean Marc / Gauchotte, Guillaume / Vogin, Guillaume

    Radiation research

    2020  Volume 193, Issue 5, Page(s) 471–480

    Abstract: Stereotactic radiotherapy (SRT) is recommended for treatment of brain oligometastasis (BoM) in patients with controlled primary disease. Where contrast enhancement enlargement occurs during follow-up, distinguishing between radionecrosis and progression ... ...

    Abstract Stereotactic radiotherapy (SRT) is recommended for treatment of brain oligometastasis (BoM) in patients with controlled primary disease. Where contrast enhancement enlargement occurs during follow-up, distinguishing between radionecrosis and progression presents a critical challenge. Without pathological confirmation, decision-making may be inappropriate and delayed. Quantitative imaging features extracted from routinely performed examinations are of interest in potentially addressing this problem. We explored the added value of the radiomics method for the differential diagnosis of these two entities. Twenty patients who received SRT for BoM, from any primary location, were included (8 radionecrosis, 12 progressions, pathologically confirmed). We assessed the clinical relevance of 1,766 radiomics features, extracted using IBEX software, from the first T1-weighted postcontrast magnetic resonance imaging (MRI) after SRT showing a lesion modification. We evaluated seven feature-selection methods and 12 classification methods in terms of respective predictive performance. The classification accuracy was measured using Cohen's kappa after leave-one-out cross-validation. In this work, the best predictive power reached was a Cohen's kappa of 0.68 (overall accuracy of 85%), expressing a strong agreement between the algorithm prediction and the histological gold standard. Prediction accuracy was 75% for radionecrosis, and 91% for progression. The area under a curve reached 0.83 using a bagging algorithm trained with the chi-square score features set. These findings indicated that the radiomics method is able to discriminate radionecrosis from progression in an accurate, early and noninvasive way. This promising study is a proof of concept, preceding a larger prospective study for defining a robust model to support decision-making in BoM. In summary, distinguishing between radionecrosis and progression is challenging without pathology. We built a classification model based on imaging data and machine learning. Using this model, we were able predict progression and radionecrosis in, respectively, 91% and 75% of cases.
    MeSH term(s) Adult ; Aged ; Brain Neoplasms/diagnosis ; Brain Neoplasms/pathology ; Brain Neoplasms/radiotherapy ; Brain Neoplasms/secondary ; Diagnosis, Differential ; Disease Progression ; Female ; Humans ; Image Processing, Computer-Assisted ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Necrosis ; Radiosurgery ; Retrospective Studies
    Language English
    Publishing date 2020-03-11
    Publishing country United States
    Document type Comparative Study ; Journal Article
    ZDB-ID 80322-4
    ISSN 1938-5404 ; 0033-7587
    ISSN (online) 1938-5404
    ISSN 0033-7587
    DOI 10.1667/RR15517.1
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    In stock of ZB MED Cologne/Königswinter

    Ue I Zs.275: Show issues Location:
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    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  9. Article ; Online: Implication of repeat insertion domains in the trans-activity of the long non-coding RNA ANRIL.

    Alfeghaly, Charbel / Sanchez, Aymeric / Rouget, Raphael / Thuillier, Quentin / Igel-Bourguignon, Valérie / Marchand, Virginie / Branlant, Christiane / Motorin, Yuri / Behm-Ansmant, Isabelle / Maenner, Sylvain

    Nucleic acids research

    2021  Volume 49, Issue 9, Page(s) 4954–4970

    Abstract: Long non-coding RNAs have emerged as critical regulators of cell homeostasis by modulating gene expression at chromatin level for instance. Here, we report that the lncRNA ANRIL, associated with several pathologies, binds to thousands of loci dispersed ... ...

    Abstract Long non-coding RNAs have emerged as critical regulators of cell homeostasis by modulating gene expression at chromatin level for instance. Here, we report that the lncRNA ANRIL, associated with several pathologies, binds to thousands of loci dispersed throughout the mammalian genome sharing a 21-bp motif enriched in G/A residues. By combining ANRIL genomic occupancy with transcriptomic analysis, we established a list of 65 and 123 genes potentially directly activated and silenced by ANRIL in trans, respectively. We also found that Exon8 of ANRIL, mainly made of transposable elements, contributes to ANRIL genomic association and consequently to its trans-activity. Furthermore, we showed that Exon8 favors ANRIL's association with the FIRRE, TPD52L1 and IGFBP3 loci to modulate their expression through H3K27me3 deposition. We also investigated the mechanisms engaged by Exon8 to favor ANRIL's association with the genome. Our data refine ANRIL's trans-activity and highlight the functional importance of TEs on ANRIL's activity.
    MeSH term(s) DNA/chemistry ; DNA Transposable Elements ; Exons ; Gene Expression Regulation ; Genetic Loci ; Genome, Human ; HEK293 Cells ; Histones/metabolism ; Humans ; RNA/chemistry ; RNA, Long Noncoding/chemistry ; RNA, Long Noncoding/metabolism
    Chemical Substances CDKN2B antisense RNA, human ; DNA Transposable Elements ; Histones ; RNA, Long Noncoding ; RNA (63231-63-0) ; DNA (9007-49-2)
    Language English
    Publishing date 2021-04-15
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkab245
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    In stock of ZB MED Cologne/Königswinter

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  10. Article ; Online: Probing small non-coding RNAs structures.

    Philippe, Jean-Vincent / Ayadi, Lilia / Branlant, Christiane / Behm-Ansmant, Isabelle

    Methods in molecular biology (Clifton, N.J.)

    2015  Volume 1296, Page(s) 119–136

    Abstract: The diverse roles of RNAs depend on their ability to fold so as to form biologically functional structures. Thus, understanding the function of a given RNA molecule often requires experimental analysis of its secondary structure by in vitro RNA probing, ... ...

    Abstract The diverse roles of RNAs depend on their ability to fold so as to form biologically functional structures. Thus, understanding the function of a given RNA molecule often requires experimental analysis of its secondary structure by in vitro RNA probing, which is more accurate than using prediction programs only. This chapter presents in vitro RNA probing protocols that we routinely use, from RNA transcript production and purification to RNA structure determination using enzymatic (RNases T1, T2, and V1) and chemical (DMS, CMCT, kethoxal, and Pb(2+)) probing performed on both unlabeled and end-labeled RNAs.
    MeSH term(s) In Vitro Techniques ; Molecular Probe Techniques ; Nucleic Acid Conformation ; RNA, Small Untranslated/chemistry
    Chemical Substances RNA, Small Untranslated
    Language English
    Publishing date 2015
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-2547-6_12
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