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Related Experiment Video

Updated: Oct 31, 2025

Identification of Circular RNAs using RNA Sequencing
08:25

Identification of Circular RNAs using RNA Sequencing

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Protocol to analyze circulating small non-coding RNAs by high-throughput RNA sequencing from human plasma samples.

Giuseppina E Grieco1,2, Guido Sebastiani1,2,3, Daniela Fignani1,2

  • 1Diabetes Unit, Department of Medicine, Surgery and Neurosciences, University of Siena, Siena 53100, Italy.

STAR Protocols
|June 30, 2021
PubMed
Summary

Developing a standardized protocol for analyzing circulating small non-coding RNAs (sncRNAs) in plasma is crucial for reliable disease biomarker discovery. This method ensures reproducible results, overcoming challenges in sncRNA detection and expression analysis.

Keywords:
High Throughput ScreeningMolecular BiologyRNAseqSequence analysisSequencing

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genomics

Background:

  • Circulating small non-coding RNAs (sncRNAs) show promise as biomarkers for disease diagnosis and treatment monitoring.
  • Significant challenges exist in identifying and validating these sncRNAs due to pre-analytical variables and diverse analytical methods.
  • Variability in sample handling and analysis leads to inconsistent study results and hinders clinical translation.

Purpose of the Study:

  • To establish a systematic and standardized protocol for the reproducible analysis of circulating sncRNAs.
  • To address the impact of pre-analytical variables on sncRNA stability and detection.
  • To provide a reliable method for high-throughput sncRNA sequencing and validation in human plasma.

Main Methods:

  • Development of a standardized protocol for processing 200 μL human plasma samples.
  • Application of high-throughput sncRNA sequencing for comprehensive analysis.
  • Validation of findings using quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Main Results:

  • The proposed protocol enables reproducible analysis of circulating sncRNAs.
  • Standardization mitigates discrepancies caused by pre-analytical factors like hemolysis and storage.
  • The method allows for reliable detection and expression profiling of sncRNAs.

Conclusions:

  • A standardized protocol is essential for the reliable identification and validation of circulating sncRNA biomarkers.
  • This reproducible method facilitates accurate disease diagnosis, staging, and therapy response assessment.
  • The protocol supports the advancement of sncRNA-based diagnostics and therapeutics.