Impact of delay to cryopreservation on RNA integrity and genome-wide expression profiles in resected tumor samples

PLoS One. 2013 Nov 20;8(11):e79826. doi: 10.1371/journal.pone.0079826. eCollection 2013.

Abstract

The quality of tissue samples and extracted mRNA is a major source of variability in tumor transcriptome analysis using genome-wide expression microarrays. During and immediately after surgical tumor resection, tissues are exposed to metabolic, biochemical and physical stresses characterized as "warm ischemia". Current practice advocates cryopreservation of biosamples within 30 minutes of resection, but this recommendation has not been systematically validated by measurements of mRNA decay over time. Using Illumina HumanHT-12 v3 Expression BeadChips, providing a genome-wide coverage of over 24,000 genes, we have analyzed gene expression variation in samples of 3 hepatocellular carcinomas (HCC) and 3 lung carcinomas (LC) cryopreserved at times up to 2 hours after resection. RNA Integrity Numbers (RIN) revealed no significant deterioration of mRNA up to 2 hours after resection. Genome-wide transcriptome analysis detected non-significant gene expression variations of -3.5%/hr (95% CI: -7.0%/hr to 0.1%/hr; p = 0.054). In LC, no consistent gene expression pattern was detected in relation with warm ischemia. In HCC, a signature of 6 up-regulated genes (CYP2E1, IGLL1, CABYR, CLDN2, NQO1, SCL13A5) and 6 down-regulated genes (MT1G, MT1H, MT1E, MT1F, HABP2, SPINK1) was identified (FDR <0.05). Overall, our observations support current recommendation of time to cryopreservation of up to 30 minutes and emphasize the need for identifying tissue-specific genes deregulated following resection to avoid misinterpreting expression changes induced by warm ischemia as pathologically significant changes.

MeSH terms

  • Calcium-Binding Proteins / genetics
  • Cell Line, Tumor
  • Cryopreservation / methods*
  • Cytochrome P-450 CYP2E1 / genetics
  • Gene Expression Profiling
  • Humans
  • In Vitro Techniques
  • Metallothionein / genetics
  • Phosphoproteins / genetics
  • RNA / genetics*
  • Serine Endopeptidases / genetics

Substances

  • CABYR protein, human
  • Calcium-Binding Proteins
  • MT1E protein, human
  • MT1F protein, human
  • MT1G protein, human
  • Phosphoproteins
  • RNA
  • Metallothionein
  • Cytochrome P-450 CYP2E1
  • HABP2 protein, human
  • Serine Endopeptidases

Grants and funding

The authors have no support or funding to report.