<ol><li>Addepalli, B., Venus, S., Thakur, P., Limbach, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+ribonuclease+activity+of+cusativin+from+Cucumis+sativus+for+mapping+nucleoside+modifications+in+RNA%5BTitle%5D)+AND+%22Analytical+and+Bioanalytical+Chemistry%22%5BJournal%5D)">Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.</a> Analytical and Bioanalytical Chemistry. 409 (24), 5645-5654 (2017).</li>
<li>Gao, H., Liu, Y., Rumley, M., Yuan, H., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequence+confirmation+of+chemically+modified+RNAs+using+exonuclease+digestion+and+matrix-assisted+laser+desorption%2Fionization+time-of-flight+mass+spectrometry%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 23 (21), 3423-3430 (2009).</li>
<li>McLuckey, S. A., Van Berkel, G. J., Glish, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tandem+mass+spectrometry+of+small%2C+multiply+charged+oligonucleotides%5BTitle%5D)+AND+%22Journal+of+The+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">Tandem mass spectrometry of small, multiply charged oligonucleotides.</a> Journal of The American Society for Mass Spectrometry. 3 (1), 60-70 (1992).</li>
<li>Fountain, K. J., Gilar, M., Gebler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+native+and+chemically+modified+oligonucleotides+by+tandem+ion-pair+reversed-phase+high-performance+liquid+chromatography%2Felectrospray+ionization+mass+spectrometry%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 17 (7), 646-653 (2003).</li>
<li>Taucher, M., Breuker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+modified+RNA+by+top-down+mass+spectrometry%5BTitle%5D)+AND+%22Angewandte+Chemie+International+Edition+in+English%22%5BJournal%5D)">Characterization of modified RNA by top-down mass spectrometry.</a> Angewandte Chemie International Edition in English. 51 (45), 11289-11292 (2012).</li>
<li>Kellner, S., Burhenne, J., Helm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+RNA+modifications%5BTitle%5D)+AND+%22RNA+Biology%22%5BJournal%5D)">Detection of RNA modifications.</a> RNA Biology. 7 (2), 237-247 (2010).</li>
<li>Thomas, B., Akoulitchev, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+of+RNA%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Mass spectrometry of RNA.</a> Trends in Biochemical Sciences. 31 (3), 173-181 (2006).</li>
<li>Bjorkbom, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bidirectional+direct+sequencing+of+noncanonical+RNA+by+two-dimensional+analysis+of+mass+chromatograms%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Bidirectional direct sequencing of noncanonical RNA by two-dimensional analysis of mass chromatograms.</a> Journal of the American Chemical Society. 137 (45), 14430-14438 (2015).</li>
<li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+general+LC-MS-based+RNA+sequencing+method+for+direct+analysis+of+multiple-base+modifications+in+RNA+mixtures%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.</a> Nucleic Acids Research. 47 (20), 125(2019).</li>
<li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+sequencing+of+tRNA+by+2D-HELS-AA+MS+Seq+reveals+its+different+isoforms+and+dynamic+base+modifications%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">Direct sequencing of tRNA by 2D-HELS-AA MS Seq reveals its different isoforms and dynamic base modifications.</a> ACS Chemical Biology. 15 (6), 1464-1472 (2020).</li>
<li>Bakin, A., Ofengand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four+newly+located+pseudouridylate+residues+in+Escherichia+coli+23S+ribosomal+RNA+are+all+at+the+peptidyltransferase+center%3A+analysis+by+the+application+of+a+new+sequencing+technique%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique.</a> Biochemistry. 32 (37), 9754-9762 (1993).</li>
<li>Cantara, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+Modification+Database%2C+RNAMDB%3A+2011+update%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">The RNA Modification Database, RNAMDB: 2011 update.</a> Nucleic Acids Research. 39 (Database issue), D195-D201 (2011).</li>
</ol>

<table><tbody><tr><td>5&#039; DNA Adenylation kit</td><td>New England Biolabs</td><td>E2610S</td><td>50uM concentration</td></tr><tr><td>6550 Q-TOF mass spectrometer</td><td>Agilent Technologies</td><td>5991-2116EN</td><td>Coupled to a 1290 Infinity LC system</td></tr><tr><td>A(5&amp;acute;)pp(5&amp;acute;)Cp-TEG-biotin-3&amp;acute;</td><td>ChemGenes</td><td>91718</td><td>HPLC purified</td></tr><tr><td>ATP&amp;gamma;S</td><td>Sigma-Aldrich</td><td>11162306001</td><td>Lithium salt</td></tr><tr><td>Bicine</td><td>Sigma-Aldrich</td><td>B8660</td><td>BioXtra, &amp;ge;99% (titration)</td></tr><tr><td>Biotin maleimide</td><td>Vector Laboratories</td><td>SP-1501</td><td>Long arm</td></tr><tr><td>C18 column</td><td>Waters</td><td>186003532</td><td>50 mm &amp;times; 2.1 mm Xbridge C18 column with a particle size of 1.7 &amp;mu;m</td></tr><tr><td>Centrifugal Vacuum Concentrator</td><td>Labconco</td><td>Refrig 115v/60hz 7310022</td><td>Labconco CentriVap</td></tr><tr><td>ChemBioDraw</td><td>PerkinElmer</td><td>ChemDraw Prime</td><td>Generate a chemical structure and property data of structures &amp;amp; fragments</td></tr><tr><td>CMC (N-cyclohexyl-N?-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate)</td><td>Sigma-Aldrich</td><td>2491-17-0</td><td>95% Purifiy</td></tr><tr><td>Cyanine3 maleimide (Cy3)</td><td>Lumiprobe</td><td>11080</td><td>Water insoluble</td></tr><tr><td>DEPC-treated water</td><td>Thermo Fisher Scientific</td><td>AM9906</td><td>Autoclaved, certified nuclease-free</td></tr><tr><td>Diisopropylamine (DIPA)</td><td>Thermo Fisher Scientific</td><td>108-18-9</td><td>99% Alfa Aesar</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855</td><td>Anhydrous dimethyl sulfoxide, 99.9%</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td>Anhydrous, crystalline, BioReagent, suitable for cell culture</td></tr><tr><td>Formic acid</td><td>Merck</td><td>64-18-6</td><td>98-100%, ACS reag, Ph Eur</td></tr><tr><td>Hexafluoro-2-propanol (HFIP)</td><td>Thermo Fisher Scientific</td><td>920-66-1</td><td>99% Acros Organics</td></tr><tr><td>LC-MS sample vials</td><td>Thermo Fisher Scientific</td><td>C4000-11</td><td>Plastic screw thread vials</td></tr><tr><td>LC-MS vial caps</td><td>Thermo Fisher Scientific</td><td>C5000-54A</td><td>Autosampler vial screw thread caps</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt; buffer</td><td>Sigma-Aldrich</td><td>88975</td><td>BioUltra, &gt;0.1 M Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;, &gt;0.2 M NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Oligo Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4060</td><td>Spin column</td></tr><tr><td>OriginLab</td><td>OriginLab</td><td>OriginPro</td><td>Data analysis and graphing software</td></tr><tr><td>pCp-biotin</td><td>TriLink BioTechnologies</td><td>NU-1706-BIO</td><td>20 ul (1 mM)</td></tr><tr><td>RNA #1--#6</td><td>Integrated DNA Technologies</td><td>Custom RNA oligos</td><td>19nt-21nt single-stranded RNAs, used without further purification</td></tr><tr><td>Rocking platform shaker</td><td>VWR</td><td>Orbital Shaker Standard 1000</td><td>Speed Range 40 to 300 rpm</td></tr><tr><td>Streptavidin magnetic beads</td><td>Thermo Fisher Scientific</td><td>88816</td><td>Binding approx. 55ug biotinylated rabbit lgG per mg of beads</td></tr><tr><td>Sulfonated Cyanine3 maleimide</td><td>Lumiprobe</td><td>11380</td><td>Water soluble</td></tr><tr><td>T4 DNA ligase 1</td><td>New England Biolabs</td><td>M0202S</td><td>400 units/uL</td></tr><tr><td>T4 polynucleotide kinase</td><td>Sigma-Aldrich</td><td>T4PNK-RO</td><td>From phage T4 am N81 pse T1 infected Escherichia coli BB</td></tr><tr><td>Tris-HCl buffer</td><td>Sigma-Aldrich</td><td>T6455</td><td>Tris-HCl Buffer, pH 10, 10&amp;times;, Antigen Retriever</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>81871</td><td>Urea for synthesis. CAS No. 57-13-6, EC Number 200-315-5.</td></tr></tbody></table>

2d-hels ms seq: a general lc-ms-based method for direct and de novo sequencing of rna mixtures with different nucleotide modifications

Watch this Scientific Journal Video about 2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications at JoVE.com

<meta charset="utf8"></head><body>2D-HELS MS Seq: Um método geral baseado em LC-MS para sequenciamento direto e de novo de misturas de RNA com diferentes modificações de nucleotídeos

2D-HELS MS Seq: Um método geral baseado em LC-MS para sequenciamento direto e de novo de misturas de RNA com diferentes modificações de nucleotídeos

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

2d-hels-ms-seq-general-lc-ms-based-method-for-direct-de-novo

Research

JoVE Journal

Genetics

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

1.9K visualizações.  New York Institute of Technology. Aqui, descrevemos um protocolo detalhado para um método de sequenciamento baseado em LC-MS que pode ser usado como um método direto para sequenciar RNA curto (

Vídeo: 2D-HELS MS Seq: Um método geral baseado em LC-MS para sequenciamento direto e de novo de misturas de RNA com diferentes modificações de nucleotídeos

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

Um método para medir RNA<em&gt; N</em<sup&gt; 6</sup&gt; -methyladenosine modificações nas células e tecidos

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Bioquímica

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Preparação da amostra para identificação de espectrometria de massa-baseada das regiões de ligação de RNA

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Melhorando O RNA-Seq pequeno: menos polarização e melhor deteção de 2 '-O-methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Genética

Analisando a dinâmica do tRNA usando AQRNA-seq

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

Autor em destaque: Papel do AQRNA-seq no mapeamento de pequenos RNAs e desvendamento de mecanismos de tradução de proteínas

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

AQRNA-seq para quantificar RNAs pequenos

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Um método rápido de alta capacidade para Ribonucleoproteínas Mapping (RNPs) no pré-mRNA Humanos

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biologia

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Caracterizando modificações de RNA em neurônios únicos usando espectrometria de massa

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

Química

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

Sondando a estrutura do RNA com perfil mutacional de sulfato de dimetilo com sequenciamento in vitro e em células

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

Uma abordagem não-sequencial para a detecção rápida da edição de RNA

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...

2D-HELS MS Seq: Um método geral baseado em LC-MS para sequenciamento direto e de novo de misturas de RNA com diferentes modificações de nucleotídeos

Resumo

Explore mais vídeos

Um método para medir RNA

Preparação da amostra para identificação de espectrometria de massa-baseada das regiões de ligação de RNA

Melhorando O RNA-Seq pequeno: menos polarização e melhor deteção de 2 '-O-methyl RNAs

AQRNA-seq para quantificar RNAs pequenos

AQRNA-seq para quantificar RNAs pequenos

AQRNA-seq para quantificar RNAs pequenos

Um método rápido de alta capacidade para Ribonucleoproteínas Mapping (RNPs) no pré-mRNA Humanos

Caracterizando modificações de RNA em neurônios únicos usando espectrometria de massa

Sondando a estrutura do RNA com perfil mutacional de sulfato de dimetilo com sequenciamento in vitro e em células

Uma abordagem não-sequencial para a detecção rápida da edição de RNA