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Related Experiment Video

Updated: May 27, 2025

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms
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Construction of RNA reference materials for improving the quantification of transcriptomic data.

Ying Yu1, Wanwan Hou1, Qingwang Chen1

  • 1State Key Laboratory of Genetic Engineering, Human Phenome Institute and School of Life Sciences, Fudan University, Shanghai, China.

Nature Protocols
|February 18, 2025
PubMed
Summary

Establishing robust RNA reference materials and ratio-based datasets is crucial for accurate RNA sequencing. This protocol ensures reliable data normalization and quality control across diverse clinical applications.

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Area of Science:

  • Molecular Biology
  • Bioinformatics
  • Genomics

Background:

  • RNA reference materials are essential for accurate RNA sequencing data normalization.
  • Conventional RNA reference datasets face limitations due to variability across batches, methods, and platforms.
  • A novel ratio-based method offers more reliable gene expression values than absolute measurements.

Purpose of the Study:

  • To describe a comprehensive, step-by-step protocol for establishing RNA reference materials and ratio-based reference datasets.
  • To detail the three key stages: reference material preparation, ratio-based dataset generation, and application.
  • To enable improved RNA sequencing data quality in clinical settings.

Main Methods:

  • Reference material preparation, homogeneity, and stability testing.
  • Construction of ratio-based reference datasets, including characterization, uncertainty estimation, and validation.
  • Application of reference materials for performance metrics, proficiency testing, and batch effect correction.

Main Results:

  • The developed protocol led to the establishment of the Quartet RNA reference materials and datasets.
  • These materials have been approved as the first nationally certified RNA reference materials in China.
  • The protocol provides a framework for generating reliable RNA reference materials and datasets.

Conclusions:

  • The described protocol offers a standardized approach to creating and utilizing RNA reference materials and datasets.
  • This method enhances the reliability and comparability of RNA sequencing data, particularly in clinical research.
  • The established national certified RNA reference materials will significantly improve RNA sequencing data quality and diagnostic accuracy.