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Title:
Efficient 5' Cap-Dependent RNA Purification: Use in Identifying and Studying Subsets of RNA | SpringerLink
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Microarray-based screening technologies have revealed a larger than expected diversity of gene expression profiles for many cells, tissues, and organisms. The complexity of RNA species, defined by their molecular structure, represents a major new development in...
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post-transcriptional gene regulation cap-binding protein eif4e post-transcriptional regulation springer science+business media micro-rna genes mir15 gene expression profiles full-length cdna ends privacy choices/manage cookies karolinska university press m7g cap analogues antisense rna transcripts differential selection approaches gene expression studies + rna-interacting proteins increased binding affinity functional eif-4ehuman colorado state university disclosing hidden transcripts high-affinity variant author information authors journal finder publish rna polymerase ii high-resolution map european economic area protein synthesis artificial tethered enzyme chronic lymphocytic leukemia s4-h2 loop spivak-kroizman sepharose-coupled m7gdp nih ro1 ca063640 humana press carries genetic information mammalian exosome mediates conditions privacy policy protocol cite protocol bajak laminar shear stress nucleic acids res international university line kansas medical center usa edyta general translational repression accepting optional cookies detection methods au-rich elements tetrahymena thermophila mrnas cytoplasmic processing bodies main content log 3β inverse race-pcr
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ScholarlyArticle:
headline:Efficient 5' Cap-Dependent RNA Purification: Use in Identifying and Studying Subsets of RNA
pageEnd:160
pageStart:147
image:https://media.springernature.com/w153/springer-static/cover/book/978-1-59745-033-1.jpg
genre:
Springer Protocols
isPartOf:
name:Post-Transcriptional Gene Regulation
isbn:
978-1-59745-033-1
978-1-58829-783-9
type:Book
publisher:
name:Humana Press
logo:
url:https://www.springernature.com/app-sn/public/images/logo-springernature.png
type:ImageObject
type:Organization
author:
name:Edyta Z. Bajak
affiliation:
name:University of Kansas Medical Center
address:
name:Department of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, USA
type:PostalAddress
type:Organization
type:Person
name:Curt H. Hagedorn
affiliation:
name:University of Kansas Medical Center
address:
name:Department of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, USA
type:PostalAddress
type:Organization
type:Person
keywords:RNA purification, 5β² RNA cap, m7G cap, eIF4E, microarray, mRNA, non-coding RNA, 3β² poly(A), deadenylation, cytoplasmic processing bodies
description:Microarray-based screening technologies have revealed a larger than expected diversity of gene expression profiles for many cells, tissues, and organisms. The complexity of RNA species, defined by their molecular structure, represents a major new development in biology. RNA not only carries genetic information in the form of templates and components of the translational machinery for protein synthesis but also directly regulates gene expression as exemplified by micro-RNAs (miRNAs). Recent evidence has demonstrated that 5β² capped and 3β² polyadenylated ends are not restricted to mRNAs, but that they are also present in precursors of both miRNAs and some antisense RNA transcripts. In addition, as many as 40% of transcribed RNAs may lack 3β² poly(A) ends. In concert with the presence of a 5β² cap (m7 GpppN), the length of the 3β² poly(A) end plays a critical role in determining the translational efficiency, stability, and the cellular distribution of a specific mRNA. RNAs with short or lacking 3β² poly(A) ends, that escape isolation and amplification with oligo(dT)-based methods, provide a challenge in RNA biology and gene expression studies. To circumvent the limitations of 3β² poly(A)-dependent RNA isolation methods, we developed an efficient RNA purification system that binds the 5β² cap of RNA with a high-affinity variant of the cap-binding protein eIF4E. This system can be used in differential selection approaches to isolate subsets of RNAs, including those with short 3β² poly(A) ends that are likely targets of post-transcriptional regulation of gene expression. The length of the 3β² poly(A) ends can be defined using a rapid polymerase chain reaction (PCR)- based approach.
datePublished:2008
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978-1-59745-033-1
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name:Department of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, USA
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name:Department of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, USA
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name:Curt H. Hagedorn
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name:Department of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, USA
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name:Department of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, USA
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