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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method
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Ribonucleoprotein purification and characterization using RNA Mango.

Shanker Shyam S Panchapakesan1, Matthew L Ferguson2,3, Eric J Hayden3

  • 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

RNA (New York, N.Y.)
|July 28, 2017
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel RNA tagging system using the Mango aptamer and a fluorogenic ligand for simplified purification and characterization of RNA-protein complexes (RNPs). This method enables efficient isolation and analysis of endogenous RNPs at various scales.

Keywords:
MangoRNARNP pull-downTO1TO3desthiobiotinfluorophorepurification

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • RNA Biology

Background:

  • Characterizing RNA-protein complexes (RNPs) is crucial but challenging in modern biology.
  • Existing methods for RNP purification and analysis can be complex and time-consuming.

Purpose of the Study:

  • To develop a simplified and versatile system for purifying and characterizing endogenous RNPs.
  • To demonstrate the utility of the Mango aptamer-based system for both small-scale pull-down assays and large-scale RNP isolation.

Main Methods:

  • Integration of the Mango aptamer into specific RNAs (U1 snRNA, 6S RNA).
  • Immobilization of Mango-tagged RNPs on streptavidin supports.
  • Purification using biotin-streptavidin interaction.
  • Characterization using fluorescence-based techniques (e.g., single-molecule fluorescence cross-correlation spectroscopy).

Main Results:

  • The Mango aptamer system successfully facilitated the purification of functional U1 snRNA-associated proteins.
  • Large-scale purification of bacterial RNA polymerase holoenzyme in complex with Mango-containing 6S RNA was achieved.
  • The system allowed for real-time monitoring of RNA binding and release using dual-color fluorescence analysis.

Conclusions:

  • The Mango aptamer and fluorogenic ligand system provides an efficient and adaptable method for purifying and characterizing endogenous RNPs.
  • This approach simplifies RNP analysis across different scales, from in vitro pull-down assays to the isolation of large cellular RNP complexes.
  • The developed system offers a valuable tool for advancing the study of RNA biology and RNP function.