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MRNA, Polyadenylated

mRNA, Polyadenylated refers to messenger RNA (mRNA) molecules that have a poly(A) tail at the 3' end.
This tail, composed of multiple adenosine monophosphate (A) residues, is added post-transcriptionally and plays a crucial role in mRNA stability, translation, and nuclear export.
Polyadenylated mRNA is the predominant form found in eukaryotic cells and is essential for efficient gene expression.
Understading the characteristics and functions of mRNA polyadenylation is key for advancing research in areas such as transcriptomics, gene regulation, and mRNA-based therapeutics like vaccines.

Most cited protocols related to «MRNA, Polyadenylated»

1
Transcriptome Profiling Across Species
For the sake of reproducibility, all data sets and scripts used to generate the benchmark files are available in Supplementary Data.
Human long non-coding and protein-coding genes were obtained from the manually curated GENCODE version 24 annotation (EnsEMBL v83 corresponding to the GRCh38 human genome assembly) selecting the long non-coding gene biotypes ‘lincRNA’ and ‘antisense’, and ‘protein_coding’ for coding genes. From each of this set, 10 000 transcripts were extracted and further divided into two sets of 5000 transcripts, that are used for the learning and the testing steps, denoted HL and HT data sets, respectively. Importantly, only one transcript per locus was extracted for all biotypes in order not to create a bias by introducing two isoforms of the same gene in both the HL and HT sets. For mouse, we used the GENCODE version M4 annotation (EnsEMBL v79) and derived the learning and testing sets in the same way as for human (denoted ML and MT). Due to the lower number of GENCODE lncRNAs annotated in mouse compared to human, each file contains ∼2000 lncRNAs and 5000 mRNAs. For ‘non-model organisms’, lncRNAs belonging to the lincRNA and antisense classes (NONCODE codes 0001 and 1000, respectively) were downloaded from the latest version of the NONCODE database (NONCODE 2016) (28 (link)) while mRNAs were retrieved from the EnsEMBL database (v84). A summary of the number of mRNAs/lncRNAs per species is available in Supplementary Table S1.
Whole transcriptome sequencing of dog RNA samples (n = 20) was performed by the LUPA consortium. These biological samples, corresponding to 16 unique tissues and 7 breeds, were obtained from the ‘Cani-DNA CRB’ biobank at the University Rennes1, CNRS-IGDR, France (http://dog-genetics.genouest.org), the biobank at University of Copenhagen, Denmark, the biobank at University of Helsinky, Finland and the Vetsuisse Biobank at University of Bern, Switzerland. The dog owners consented to the use of the data for research purposes anonymously. RNAs were extracted from tissues using the NucleoSpin RNA kit (Macherey–Nagel) according to the manufacturer's instructions. Polyadenylated RNAs were captured by oligo-dT beads and RNA-seq libraries were constructed via the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. Sequencing was done in paired-end and stranded fashion on the HiSeq2000 platform using v3 chemistry (TruSeq PE Cluster Kit v3-cBot-HS, TruSeq SBS Kit v3-HS, TruSeq SR Cluster Kit v3-cBot-HS) to a depth of about 50 million reads per tissue (Supplementary Table S2). The RNA-seq data are available in the short read archive (SRA) under NCBI bioproject PRJNA327075 and SRA accession SRP077559.
Wucher V., Legeai F., Hédan B., Rizk G., Lagoutte L., Leeb T., Jagannathan V., Cadieu E., David A., Lohi H., Cirera S., Fredholm M., Botherel N., Leegwater P.A., Le Béguec C., Fieten H., Johnson J., Alföldi J., André C., Lindblad-Toh K., Hitte C, & Derrien T. (2017). FEELnc: a tool for long non-coding RNA annotation and its application to the dog transcriptome. Nucleic Acids Research, 45(8), e57.
Publication 2017
Biopharmaceuticals Breeding Gene Products, Protein Genes Genes, vif Genome, Human Homo sapiens Long Intergenic Non-Protein Coding RNA Mus oligo (dT) Protein Isoforms RNA, Long Untranslated RNA, Messenger RNA, Polyadenylated RNA-Seq Tissues
2
Preparing Macrophages for RNA-Seq Analysis

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Publication 2012
beta-Tubulin Bone Marrow Buffers Cells Chromatin Cytoplasm deoxyuridine triphosphate Detergents Endoribonucleases Histone H3 Homo sapiens Immunoblotting Lipid A Macrophage Mus Nonidet P-40 Poly A Ribosomal RNA RNA, Messenger RNA, Polyadenylated Subcellular Fractions Sucrose
3
Large-Scale RNA-seq of Cell Lines
RNA-seq and analysis were performed for 1,019 cell lines as previously described5 (link). In summary, non-strand specific RNA sequencing was performed using large-scale, automated method of the Illumina TruSeq RNA Sample Preparation protocol. Oligo-dT beads were used to select polyadenylated mRNA. The selected RNA was then heat fragmented and randomly primed before cDNA synthesis. To maximize power to detect fusions, the insert size of fragments was set to 400nt. The resultant cDNA then went through Illumina library preparation (end-repair, base ‘A’ addition, adaptor ligation, and enrichment) using Broad-designed indexed adapters for multiplexing. Sequencing was performed on the Illumina HiSeq 2000 or HiSeq 2500 instruments with sequence coverage of no less than 100 million paired 101 nucleotides-long reads per sample.
Ghandi M., Huang F.W., Jané-Valbuena J., Kryukov G.V., Lo C.C., McDonald ER I.I.I., Barretina J., Gelfand E.T., Bielski C.M., Li H., Hu K., Andreev-Drakhlin A.Y., Kim J., Hess J.M., Haas B.J., Aguet F., Weir B.A., Rothberg M.V., Paolella B.R., Lawrence M.S., Akbani R., Lu Y., Tiv H.L., Gokhale P.C., de Weck A., Mansour A.A., Oh C., Shih J., Hadi K., Rosen Y., Bistline J., Venkatesan K., Reddy A., Sonkin D., Liu M., Lehar J., Korn J.M., Porter D.A., Jones M.D., Golji J., Caponigro G., Taylor J.E., Dunning C.M., Creech A.L., Warren A.C., McFarland J.M., Zamanighomi M., Kauffmann A., Stransky N., Imielinski M., Maruvka Y.E., Cherniack A.D., Tsherniak A., Vazquez F., Jaffe J.D., Lane A.A., Weinstock D.M., Johannessen C.M., Morrissey M.P., Stegmeier F., Schlegel R., Hahn W.C., Getz G., Mills G.B., Boehm J.S., Golub T.R., Garraway L.A, & Sellers W.R. (2019). Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature, 569(7757), 503-508.
Publication 2019
Anabolism cDNA Library Cell Lines DNA, Complementary Ligation mRNA, Polyadenylated Nucleotides oligo (dT) RNA-Seq
4
Comparative Analysis of RNA-seq Datasets with Varying Quality
This study used a total of four datasets including three published RNA-seq datasets. All three published datasets were obtained from the NCBI Sequence Read Archive (SRA; http://www.ncbi.nlm.nih.gov/Traces/sra/) or Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). Sequencing reads from all samples were independently aligned to the human reference genome (hg19/GRCh37) using Tophat (v2.0.6) software configured with default options.

Human U-251 MG brain glioblastoma cell lines (GBM) [5 (link)]. This dataset has 12 pair-end RNA-seq data files available under SRA accession SRP023548. Samples in this dataset have a wide range of RIN values: three samples with RIN value of 10 (SRR873838, SRR873834 and SRR873822), two samples with RIN value of 8 (SRR879615 and SRR879800), three samples with RIN value of 6 (SRR880232, SRR881272 and SRR880070), three samples with RIN value of 4 (SRR881852, SRR881451, and SRR881672) and one sample with RIN value of 2 (SRR881985). Additional file 1: Table S1 presents the details of this dataset.

Human peripheral blood mononuclear cells (PBMC) [4 (link)]. This dataset has 20 single-end RNA-seq data files available under SRA accession SRP041955. This dataset was developed to estimate the in vitro degradation at 12 h, 24 h, 48 h and 84 h. Additional file 2: Table S2 presents the details of the samples along with their associated RIN values (varied from 2.8 to 9.4).

Sequencing quality control consortium data set (SEQC) [28 (link)]. The Sequencing Quality Control Consortium analyzed samples containing reference RNA. This dataset was downloaded from NCBI Gene Expression Omnibus (GEO) with accession number GSE49712. This SEQC subset has a total of 10 samples. Group A contains 5 replicates (SRR950078, SRR950080, SRR950082, SRR950084 and SRR950086) of the Stratagene Universal Human Reference RNA (UHRR) and Group B has 5 replicates (SRR950079, SRR950081, SRR950083, SRR950085 and SRR950087) of the Ambion Human Brain Reference RNA (HBRR). ERCC (External RNA Controls Consortium) control mix was spiked in both groups at 2 % by volume. This control mixture contains 92 synthetic polyadenylated oligonucleotides of 250-2000 nucleotides in length, which were meant to resemble human transcripts.

Human prostate cancer tissue samples (mCRPC). This study was approved by the Mayo Clinic Institutional Review Board and conducted in accordance with the Declaration of Helsinki. We obtained a total of 120 samples from 46 castration-resistant prostate cancer patients. Out of the collected 120 samples, 62 were blood samples, 18 were metastatic rib lesion biopsies and 40 were metastatic bone tissue biopsies. Tissues were snap frozen with liquid nitrogen and RNA was harvested using Rneasy Plus Mini Kit (Qiagen). RNA libraries were prepared according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). Briefly, poly-A mRNA was purified from total RNA using oligo dT magnetic beads. The purified mRNA was fragmented at 95 °C for 8 min and eluted from the beads. Double stranded cDNA was made using SuperScript III reverse transcriptase, random primers (Invitrogen, Carlsbad, CA) and DNA polymerase I and RNase H. The cDNA ends were repaired and an “A” base added to the 3′ ends. TruSeq paired end index DNA adaptors (Illumina, San Diego CA) with a single “T” base overhang at the 3′ end were ligated and the resulting constructs were purified using AMPure SPRI beads from Agencourt. The adapter-modified DNA fragments were enriched by 12 cycles of PCR using Illumina TruSeq PCR primers. The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip and Qubit fluorometry (Invitrogen, Carlsbad, CA). Pair-end RNA sequencing was performed using Illumina HiSeq 2500. Additional file 3: Table S3 presents the details of this dataset.

Wang L., Nie J., Sicotte H., Li Y., Eckel-Passow J.E., Dasari S., Vedell P.T., Barman P., Wang L., Weinshiboum R., Jen J., Huang H., Kohli M, & Kocher J.P. (2016). Measure transcript integrity using RNA-seq data. BMC Bioinformatics, 17, 58.
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Publication 2016
5
RNA-Binding Protein Interactome Capture
Initial UV crosslinking and oligo(dT) purification followed the mRNA interactome capture protocol (Castello et al., 2013b (link)). Complete proteolytic digestions were performed with LysC or ArgC for 8 hr at 37°C. Polyadenylated RNA and crosslinked peptides were diluted in 20 mM Tris-HCl, 500 mM LiCl, 1 mM DTT, and 0.5 mM EDTA and recaptured on oligo(dT) beads. The supernatant was processed for MS (released peptides). oligo(dT) beads were washed as in Castello et al. (2013b (link)). All fractions were treated with trypsin and labeled with stable isotopes in vitro (Boersema et al., 2008 (link)). Peptides were analyzed on a liquid chromatography-tandem MS (LC-MS/MS) platform. The R-scripts used for the analyses can be found in the R/Bioconductor data-package RBDmapHeLa (http://www.bioconductor.org). RBDmap data can be accessed under http://www-huber.embl.de/users/befische/RBDmap.
Castello A., Fischer B., Frese C.K., Horos R., Alleaume A.M., Foehr S., Curk T., Krijgsveld J, & Hentze M.W. (2016). Comprehensive Identification of RNA-Binding Domains in Human Cells. Molecular Cell, 63(4), 696-710.
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Publication 2016
Digestion Edetic Acid Isotopes Liquid Chromatography Oligonucleotides Peptide Hydrolases Peptides RNA, Messenger RNA, Polyadenylated Tandem Mass Spectrometry Tromethamine Trypsin

Most recents protocols related to «MRNA, Polyadenylated»

1
RNA-Seq Library Preparation and Sequencing
RNA samples were used for library preparation using an NEB Next® Ultra RNA Library Prep Kit for Illumina® Indices (New England Biolabs, MA, USA) to multiplex multiple samples. Briefly, mRNA was purified from total RNA using poly-T oligos attached to magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by a second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, and size selection. After amplification and purification, the insert size of the library was validated on an Agilent 2100 and quantified using quantitative PCR (Q-PCR). Libraries were then sequenced on an Illumina NovaSeq 6000 S4 flowcell with PE150 according to results from library quality control and expected data volume. The library preparation/sequencing/analysis were performed by Novogene (UK) Company Limited (Cambridge, UK).
Wang L., Koelink P.J., Garssen J., Folkerts G., Henricks P.A, & Braber S. (2024). Gut Microbiome and Transcriptomic Changes in Cigarette Smoke-Exposed Mice Compared to COPD and CD Patient Datasets. International Journal of Molecular Sciences, 25(7), 4058.
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Publication 2024
2
Quantifying Viral Replication in Lungs
Our RNA-seq library preparation captured both cellular and viral mRNA (positive-sense, polyadenylated viral RNA). IAV load was measured using the viral mRNA levels in the lung tissue based on the RNA-seq measurements. As detailed above, we aligned the RNA-seq reads of all samples against the mouse and the IAV genomes and quantified the viral gene expression. The reported viral load is the average of (log2-scaled) viral mRNA across all IAV genes.
Of note, since the cellular replication of IAV is characterized by the synthesis of polyadenylated mRNA molecules with positive polarity, but free IAV virions encapsidate the non-polyadenylated viral genome with a negative polarity, the quantification of the viral polyadenylated mRNAs reflects intracellular viral replication rather than free virions.
Cohn O., Yankovitz G., Mandelboim M., Peshes-Yaloz N., Brandes R., Bacharach E, & Gat-Viks I. (2024). The host transcriptional response to superinfection by influenza A virus and Streptococcus pneumoniae. mSystems, 9(4), e01048-23.
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Publication 2024
3
Yak Lung Transcriptome Profiling
We extracted total RNA from the frozen yak lung tissue samples from each age group by using the TRIzol (Invitrogen, CA, United States) reagent. The extracted RNA samples were assessed for integrity and potential contamination through 1% agarose gel electrophoresis. RNA purity was determined on a NanoPhotometer spectrophotometer by measuring the OD260/280 and OD260/230 ratios. Moreover, RNA integrity was verified on an Agilent 2100 bioanalyzer, and rRNA was removed. Polyadenylated eukaryotic mRNA was then enriched using magnetic beads with Oligo(dT)s. Subsequently, the enriched polyadenylated mRNA was fragmented through ultrasonication. The fragmented mRNA was used as a template for double-stranded complementary DNA (cDNA) synthesis. The resulting polymerase chain reaction (PCR) products (i.e., the cDNA) were then subjected to selection, amplification, and purification by using AMPure XP beads. Finally, the purified cDNA was used to construct cDNA libraries. This process ensured. high-quality RNA extraction and polyadenylated mRNA enrichment, followed by generation of representative cDNA libraries for subsequent analyses. The results provided crucial insights into lung tissue transcriptomes across different age groups of yak.
Xie X., Wei Y., Cui Y., Zhang Q., Lu H., Chen L, & He J. (2024). Transcriptomics reveals age-related changes in ion transport–related factors in yak lungs. Frontiers in Veterinary Science, 11, 1374794.
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Publication 2024
4
Training mRNA and mtRNA Models
For the development of the mRNA and mtRNA models, MinION DRS signals from human heart as well as GM12878, GM24385, HEK293, HeLa, KOPN8 and REH cell lines (sources and catalog numbers listed below) were used for training and evaluating the mRNA and mtRNA models (Suppl. Table 6). The total RNA of the GM24385 cell line was in vitro polyadenylated (as described above – RISER model development) prior to sequencing to include RNAs that are not natively polyadenylated in the training data.
Sneddon A., Ravindran A., Shanmuganandam S., Kanchi M., Hein N., Jiang S., Shirokikh N, & Eyras E. (2024). Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER. Nature Communications, 15, 4422.
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Publication 2024
5
Spleen Total RNA Extraction and Sequencing
Total RNA was isolated from spleen using Trizol reagent. Then mRNA samples library construction and high-throughput sequencing were performed by Hangzhou KaiTai Biotechnology Co., Ltd. In brief, Polyadenylated RNA was enriched from total RNA. mRNA samples were fragmented into 300-nucleotide-long fragments using RNA Fragmentation Reagents (Ambion, AM8740) and used for library construction and performed high-throughput sequencing on Illumina Novaseq 6000.
Liao C., Luo S., Liu X., Zhang L., Xie P., Zhou W., Lu Y., Zhong H., Zhang X., Xiong Z., Huang X., Mo G., Ma D, & Tang J. (2024). Siglec-F+ neutrophils in the spleen induce immunosuppression following acute infection. Theranostics, 14(6), 2589-2604.
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Publication 2024

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More about "MRNA, Polyadenylated"

Messenger RNA (mRNA) is a crucial molecule in gene expression, responsible for carrying the genetic information from the nucleus to the ribosomes where proteins are synthesized.
Polyadenylation is a crucial post-transcriptional modification that involves the addition of a poly(A) tail to the 3' end of mRNA molecules.
This tail, composed of multiple adenosine monophosphate (A) residues, plays a vital role in mRNA stability, translation, and nuclear export.
Understanding the characteristics and functions of mRNA polyadenylation is essential for advancing research in areas such as transcriptomics, gene regulation, and mRNA-based therapeutics like vaccines.
Techniques like TRIzol reagent, TRIzol, and RNeasy Mini Kit are commonly used for RNA extraction and purification, while the Dynabeads mRNA Purification Kit is used for specific isolation of polyadenylated mRNA.
High-throughput sequencing platforms like the HiSeq 2500 and HiSeq 2000 are used for transcriptome analysis, and the Poly(A) Tailing Kit and SMART-RACE kit are used for 3' end modification and amplification of mRNA, respectively.
The Agilent 2100 Bioanalyzer is a valuable tool for quality control and quantification of RNA samples.
PubCompare.ai, an AI-driven platform, enhances the reproducibility and accuracy of mRNA research by helping researchers locate the best protocols from literature, pre-prints, and patents using intelligent comparisons.
This tool can optimize mRNA research by identifying the most effective products and procedures, even with a typo in the text.
By leveraging the insights gained from the MeSH term description and the Metadescription, researchers can advance their understanding of mRNA polyadenylation and its applications in various fields, including gene expression, therapeutics, and diagnostics.