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Analyzing Multifactorial RNA-Seq Experiments with DiCoExpress
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Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling.

K S Keslar1, M Lin, A A Zmijewska

  • 1Department of Immunology, Cleveland Clinic, Cleveland, OH, USA. keslark@ccf.org

American Journal of Transplantation : Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons
|June 28, 2013
PubMed
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Standardized gene expression analysis in transplant recipients yields consistent results across multiple labs. This validated method enables reliable monitoring of graft function using blood and urine samples.

Area of Science:

  • Biomarkers
  • Molecular Diagnostics
  • Translational Research

Background:

  • Gene expression profiling of blood and urine shows promise for monitoring transplant graft function.
  • Current diverse protocols hinder data sharing and inter-laboratory comparison.

Purpose of the Study:

  • To evaluate the performance of RNA isolation, reverse transcription, and quantitative polymerase chain reaction (qPCR) methods.
  • To determine if a standardized protocol yields consistent results across multiple research centers.

Main Methods:

  • A standardized protocol for RNA isolation, reverse transcription, and qPCR was distributed to six participating sites.
  • Specific genes (18S, PRF, GZB, IL8, CXCL9, CXCL10) were analyzed in blood and urine samples.
  • All data were centralized for analysis.

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Main Results:

  • All sites demonstrated proficiency in RNA isolation and qPCR.
  • Gene expression measurements showed high correlations (>0.938) across all targets and samples.
  • The coefficient of variation for fold-changes was <40%, and control samples were quantified accurately (within a factor of 1.5).

Conclusions:

  • A validated, standardized method for gene expression measurement in blood and urine can achieve consistent results across multiple laboratories.
  • This approach facilitates reliable graft function monitoring in transplant recipients.
  • The established protocol supports collaborative research and data standardization in the field.