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Related Concept Videos

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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Author Spotlight: Exploring the Frontier of mRNA Research with Poly A Tail Analysis Techniques
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Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing.

Koichi Ogami1, Yuka Oishi2, Shin-Ichi Hoshino2

  • 1Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 Japan; Division of Molecular Oncology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

STAR Protocols
|May 27, 2023
PubMed
Summary

This study introduces a nanopore sequencing method to accurately measure mRNA poly(A) tail length. This technique enables comprehensive analysis of gene expression, RNA splicing, and modifications.

Keywords:
Cell BiologyMolecular Biology

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Poly(A) tail metabolism is crucial for post-transcriptional gene expression regulation.
  • Accurate measurement of poly(A) tail length is essential for understanding gene expression dynamics.

Purpose of the Study:

  • To present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing.
  • To exclude truncated RNAs from poly(A) tail length measurements.

Main Methods:

  • Protocol development for analyzing intact mRNA poly(A) tail length.
  • Utilizes nanopore direct RNA sequencing.
  • Includes steps for recombinant eIF4E mutant protein preparation, m7G-capped RNA purification, library preparation, and sequencing.

Main Results:

  • The protocol enables accurate measurement of intact mRNA poly(A) tail length.
  • Resulting data supports expression profiling and poly(A) tail length estimation.
  • The method also facilitates detection of alternative splicing, polyadenylation events, and RNA base modifications.

Conclusions:

  • This protocol provides a robust method for analyzing mRNA poly(A) tail length.
  • The technique offers a comprehensive approach to studying various aspects of RNA biology.
  • The method is valuable for advancing research in gene expression regulation.