ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology

PLoS One. 2016 Nov 1;11(11):e0165181. doi: 10.1371/journal.pone.0165181. eCollection 2016.


Background: Neuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes.

Material and methods: Forty-three representative FFPE-specimens were used for mRNA expression analysis using the digital nCounter technology (NanoString). Based on recent literature, a total of 91 mRNA targets were investigated as potential tumor markers or reference genes. The geNorm, NormFinder algorithms and coefficient of correlation were used to identify the most stable reference genes. Statistical analysis was performed by using the R programming environment (version 3.1.1).

Results: RNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/μl. However, the nCounter technology gave evaluable results for all samples tested. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation.

Conclusion: FFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP are potent reference genes in neuroendocrine tumors of the lung.

MeSH terms

  • Cyclin-Dependent Kinase Inhibitor p27 / genetics*
  • GRB2 Adaptor Protein / genetics*
  • Gene Expression / genetics
  • Gene Expression Regulation, Neoplastic / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / genetics*
  • Humans
  • Lung Neoplasms / genetics*
  • Neuroendocrine Tumors / genetics*
  • Paraffin Embedding / methods
  • RNA, Messenger / genetics
  • Syntenins / genetics*
  • Tissue Fixation / methods
  • rhoA GTP-Binding Protein / genetics*


  • CDKN1B protein, human
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • RNA, Messenger
  • SDCBP protein, human
  • Syntenins
  • RHOA protein, human
  • Cyclin-Dependent Kinase Inhibitor p27
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
  • rhoA GTP-Binding Protein

Grant support

The study was financed by the Institute of Pathology and Ruhrlandklinik.