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检索条件"主题词=Splicing Model Analysis"

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Single-cell mRNA quantification and differential analysis with Census

Single-cell mRNA quantification and differential analysis wi...

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作者： Xiaojie Qiu Andrew Hill Jonathan Packer Dejun Lin Yi-An Ma Cole Trapnell

Single-cell gene expression studies promise to reveal rare cell types and cryptic states, but the high variability of single-cell RNA-seq measurements frustrates efforts to assay transcriptional differences between cells. We introduce the Census algorithm to convert relative RNA-seq expression levels into relative transcript counts without the need for experimental spike-in controls. Analyzing changes in relative transcript counts led to dramatic improvements in accuracy compared to normalized read counts and enabled new statistical tests for identifying developmentally regulated genes. Census counts can be analyzed with widely used regression techniques to reveal changes in cell-fate-dependent gene expression, splicing patterns and allelic imbalances. We reanalyzed single-cell data from several developmental and disease studies, and demonstrate that Census enabled robust analysis at multiple layers of gene regulation. Census is freely available through our updated single-cell analysis toolkit, Monocle 2. less

关键词： Single-cell RNA-seq Statistical Calculation Cell Lysis Library Preparation splicing model analysis Cufflinks Computational biology and bioinformatics Biotechnology

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Bioinformatic analysis of Chloroplast Gene Expression and RNA Posttranscriptional Maturations Using RNA Sequencing

Bioinformatic Analysis of Chloroplast Gene Expression and RN...

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作者： Bastien Malbert Guillem Rigaill Veronique Brunaud Claire Lurin Etienne Delannoy

Sequencing of total RNA enables the study of the whole plant transcriptome resulting from the simultaneous expression of the three genomes of plant cells (located in the nucleus, mitochondrion and chloroplast). While commonly used for the quantification of the nuclear gene expression, this method remains complex and challenging when applied to organellar genomes and/or when used to quantify posttranscriptional RNA maturations. Here we propose a complete bioinformatical and statistical pipeline to fully characterize the differences in the chloroplast transcriptome between two conditions. Experimental design as well as bioinformatics and statistical analyses are described in order to quantify both gene expression and RNA posttranscriptional maturations, i.e., RNA splicing, editing, and processing, and identify statistically significant differences. less

关键词： splicing model analysis RNA-seq Gene Expression analysis RNA Extraction Library Preparation Base-calling Statistical Calculation TruSeq Trimmomatic bedtools Processing splicing Editing Chloroplast transcriptome Differentially expressed

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Pre-mRNA splicing in the Nuclei of

Pre-mRNA Splicing in the Nuclei of

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作者： Kyong Hwa Moon Xinliang Zhao Yi-Tao Yu

Xenopus oocytes have been utilized in a number of laboratories as an experimental system to study a variety of biological processes. Here, we describe its application to functional studies of spliceosomal small nuclear RNAs (snRNAs) in pre-messenger RNA (pre-mRNA) splicing, a process that occurs extremely efficiently in Xenopus oocytes. A DNA oligonucleotide complementary to an snRNA of interest is injected into the oocyte cytoplasm. The oligonucleotide subsequently diffuses into the nucleus and hybridizes to the target snRNA, thereby triggering snRNA degradation via endogenous RNase H activity. By the time the endogenous snRNA is depleted, the DNA oligonucleotide itself is degraded by endogenous deoxyribonuclease (DNase) activity. In principle, this procedure enables one to quantitatively deplete any snRNA of choice. Subsequently, a rescuing snRNA that is constructed in vitro may be injected into the snRNA-depleted oocytes to restore the splicing function. After reconstitution, a radiolabeled splicing substrate is injected into the nuclei of the oocytes. These oocyte nuclei are then manually isolated and used to prepare both nuclear RNA for splicing assays and nuclear extract for spliceosome assembly assays. The ability of an injected rescuing snRNA to reconstitute splicing can therefore be tested. Because all types of rescuing snRNAs (e.g., mutant snRNAs, snRNAs with or without modified nucleotides) can be constructed readily, the results obtained from this procedure provide valuable information on the function of a particular snRNA of interest in pre-mRNA splicing. less

关键词： Microinjection Autoradiography In Vitro Transcription Dissection RNA Extraction splicing model analysis Microscopy splicing Assay Xenopus spliceosome Depletion-reconstitution nuclear isolation pre-mRNA splicing U2 snRNA

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Transcriptome Assembly and Alternative splicing analysis

Transcriptome Assembly and Alternative Splicing Analysis

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作者： Paola Bonizzoni Gianluca Della Vedova Graziano Pesole Ernesto Picardi Yuri Pirola Raffaella Rizzi

Alternative splicing (AS) is the molecular phenomenon whereby multiple transcripts are produced from the same gene locus. As a consequence, it is responsible for the expansion of eukaryotic transcriptomes. Aberrant AS is involved in the onset and progression of several human diseases. Therefore, the characterization of exon–intron structure of a gene and the detection of corresponding transcript isoforms is an extremely relevant biological task. Nonetheless, the computational prediction of AS events and the repertoire of alternative transcripts is yet a challenging issue. Hereafter we introduce PIntron, a software package to predict the exon-intron structure and the full-length isoforms of a gene given a genomic region and a set of transcripts (ESTs and/or mRNAs). The software is open source and available at http://*** . PIntron has been designed for (and extensively tested on) a standard workstation without requiring dedicated expensive hardware. It easily manages large genomic regions and more than 20,000 ESTs, achieving good accuracy as shown in an experimental evaluation performed on 112 well-annotated genes selected from the ENCODE human regions used as training set in the EGASP competition. less

关键词： splicing model analysis Sequence Annotation GenBank Python Homo sapiens Alternative splicing Spliced alignment Gene structure Expressed isoforms Software package

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ASPicDB: A Database Web Tool for Alternative splicing analysis

ASPicDB: A Database Web Tool for Alternative Splicing Analys...

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作者： Mattia D’Antonio Tiziana Castrgnanò Matteo Pallocca Anna Maria D’Erchia Ernesto Picardi Graziano Pesole

Alternative splicing (AS) is a basic molecular phenomenon that increases the functional complexity of higher eukaryotic transcriptomes. Indeed, through AS individual gene loci can generate multiple RNAs from the same pre-mRNA. AS has been investigated in a variety of clinical and pathological studies, such as the transcriptome regulation in cancer. In human, recent works based on massive RNA sequencing indicate that >95 % of pre-mRNAs are processed to yield multiple transcripts. Given the biological relevance of AS, several computational efforts have been done leading to the implementation of novel algorithms and specific specialized databases. Here we describe the web application ASPicDB that allows the recovery of detailed biological information about the splicing mechanism. ASPicDB provides powerful querying systems to interrogate AS events at gene, transcript, and protein levels. Finally, ASPicDB includes web visualization instruments to browse and export results for further off-line analyses. less

关键词： Transcriptomics splicing model analysis RNA-seq Swiss-Prot Protein Structure analysis Statistical Calculation Cross-linking GenBank Ensembl Bioinformatics Alternative splicing Transcription Database Transcriptome Web tool Alternative isoforms Transcript prediction

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Methods to Study splicing from High-Throughput RNA Sequencing Data

Methods to Study Splicing from High-Throughput RNA Sequencin...

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作者： Gael P. Alamancos Eneritz Agirre Eduardo Eyras

The development of novel high-throughput sequencing (HTS) methods for RNA (RNA-Seq) has provided a very powerful mean to study splicing under multiple conditions at unprecedented depth. However, the complexity of the information to be analyzed has turned this into a challenging task. In the last few years, a plethora of tools have been developed, allowing researchers to process RNA-Seq data to study the expression of isoforms and splicing events, and their relative changes under different conditions. We provide an overview of the methods available to study splicing from short RNA-Seq data, which could serve as an entry point for users who need to decide on a suitable tool for a specific analysis. We also attempt to propose a classification of the tools according to the operations they do, to facilitate the comparison and choice of methods.

关键词： RNA-seq High-Throughput Sequencing TopHat Bowtie Cufflinks Gene Prediction splicing model analysis Trinity Gene Expression analysis Cuffcompare Genome Visualization Microarray Quantification Reconstruction Alternative splicing splicing Isoform

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High-Resolution RT-PCR analysis of Alternative Barley Transcripts

High-Resolution RT-PCR Analysis of Alternative Barley Transc...

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作者： Craig G. Simpson John Fuller Paulo Rapazote-Flores Claus-Dieter Mayer Cristiane P. Calixto Linda Milne Pete E. Hedley Clare Booth Robbie Waugh John W. Brown

Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives confidence or otherwise to the genome-wide global changes in alternatively spliced transcripts reported by RNA-seq. This simple assay can readily be applied to perform detailed transcript isoform analysis for any gene in any species. less

关键词： RNA-seq RNA Extraction PCR Reverse Transcription PCR Statistical Calculation Capillary Electrophoresis (CE) Nucleic Acid Fragment analysis splicing model analysis RNA-Seq analysis Alternative splicing

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Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy

Peptide-Conjugated PMOs for the Treatment of Myotonic Dystro...

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作者： Jessica Stoodley David Seoane Miraz Yahya Jad Mathieu Fischer Matthew J. Wood Miguel A. Varela

Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide–PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal musclephenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCRRT-PCRsplicing analysis. less

关键词： FISH splicing model analysis Gradient Elution Sterilization Spectroscopy MALDI Cell Fixation Sample Comparison Dissection Homogenization Gel Electrophoresis Gel Imaging ImageJ FPLC Immunoassay Formaldehyde Fixation ELISA Saccharomyces cerevisiae Mus musculus Homo sapiens DM1 splicing Myotonic dystrophy Cell-penetrating peptide Muscle Antisense oligonucleotides PMO MBNL1 Anti-DIG Anti-mouse

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Identifying Target RNAs of PARPs

Identifying Target RNAs of PARPs

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作者： Florian J. Bock Paul Chang

Posttranscriptional regulation of RNA is an important component of gene expression by controlling the total amount of mRNA available for translation into protein. It involves multiple pathways including nuclear processing of mRNA and its precursors, RNA silencing, and regulation of RNA decay. Poly(ADP-ribose) polymerases (PARPs), enzymes that modify target proteins with ADP-ribose, play important roles in several RNA-regulatory pathways. RNA-binding PARPs target specific transcripts for regulation, and multiple PARPs ADP-ribosylate RNA-regulatory proteins to alter their localization, activity, or RNA binding. Additionally, RNA-binding proteins can bind directly to poly(ADP-ribose) with various effects on their function. Here we describe methods to identify and confirm specific transcripts that are regulated by PARPs. less

关键词： RNA-seq Cell And Tissue Culture Western Blot Transfection RNA Purification High-Throughput Sequencing Recombinant Protein Expression DNA Purification Electrophoresis splicing model analysis Gene Expression analysis Library Preparation RNA Extraction Genomics Real-time PCR EMSA Data analysis Homo sapiens HeLa RNA PARP ADP-ribosylation Anti-SBP

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Genome-Wide Annotation of circRNAs and Their Alternative Back-splicing/splicing with CIRCexplorer Pipeline

Genome-Wide Annotation of circRNAs and Their Alternative Bac...

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作者： Rui Dong Xu-Kai Ma Ling-Ling Chen Li Yang

Circular RNAs (circRNAs) derived from back-spliced exons were sporadically identified about 25 years ago, and have been recently re-discovered genome-wide across different species. Interestingly, one gene locus can generate multiple circRNAs through alternative back-splicing and/or alternative splicing, thus expanding our understanding on the diversity and complexity of transcriptomes. Precise annotation of circRNAs with their alternative back-splicing and alternative splicing events is the basis for the functional characterization of different categories of circRNAs. Here we describe a step-by-step computational scheme to annotate circRNAs from publicly available RNA sequencing datasets with the CIRCexplorer2 pipeline. less

关键词： Genome Visualization RNA-seq Bowtie Cufflinks splicing model analysis TopHat Python bedtools Homo sapiens Alternative splicing Computational biology CircRNA CIRCexplorer Alternative back-splicing

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