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A Modified Precipitation Method to Isolate Urinary Exosomes
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Capturing the Kidney Transcriptome by Urinary Extracellular Vesicles-From Pre-Analytical Obstacles to Biomarker

Karina Barreiro1,2, Om Prakash Dwivedi1, Antti Rannikko3,4

  • 1Institute for Molecular Medicine Finland FIMM, HiLIFE, University of Helsinki, 00290 Helsinki, Finland.

Genes
|July 29, 2023
PubMed
Summary

Urinary extracellular vesicles (uEV) contain RNA biomarkers for kidney disease. Preanalytical choices like storage temperature impact RNA levels, but stable reference mRNAs were identified for reliable uEV biomarker studies.

Keywords:
diabetic kidney diseaseexosomesmRNAmiRNAreference genessequencingurinary extracellular vesiclesurine

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genomics

Background:

  • Urinary extracellular vesicles (uEV) offer non-invasive RNA biomarkers for genitourinary diseases.
  • Lack of knowledge on reference genes and preanalytical variable effects hinders uEV biomarker studies.

Purpose of the Study:

  • To assess the impact of urine storage temperature and isolation workflow on kidney-linked RNAs in uEVs.
  • To identify stable messenger RNA (mRNA) reference genes across diverse uEV datasets for biomarker normalization.

Main Methods:

  • Analysis of nine RNA sequencing datasets from healthy individuals and patients with prostate cancer or type 1 diabetes.
  • Focus on kidney-linked RNAs, including diabetic kidney disease (DKD)-linked microRNAs (miRNAs).
  • RNA analysis using expression heatmaps, hierarchical clustering, and selection of stable mRNAs based on normalized counts and coefficient of variation.

Main Results:

  • Kidney-linked RNAs decreased after urine storage at -20 °C compared to -80 °C.
  • Different isolation workflows exhibited varying efficiencies in capturing kidney-linked RNAs.
  • Ultracentrifugation successfully isolated DKD-linked miRNAs that differentiated healthy and diabetic macroalbuminuria groups.
  • Eleven mRNAs demonstrated stable expression across all analyzed uEV datasets.

Conclusions:

  • Preanalytical choices significantly influence kidney-linked RNA detection in uEVs.
  • Analyzing kidney-linked RNAs alongside global correlation analysis can reveal subtle differences.
  • The identified stable reference mRNAs and validated DKD markers support the advancement of uEV biomarker research.