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

• Published in: PloS one 2016-11, Vol.11 (11), p.e0165181-e0165181
• Main Authors: Walter, Robert Fred Henry, Werner, Robert, Vollbrecht, Claudia, Hager, Thomas, Flom, Elena, Christoph, Daniel Christian, Schmeller, Jan, Schmid, Kurt Werner, Wohlschlaeger, Jeremias, Mairinger, Fabian Dominik
• Format: Article
• Language: English
• Subjects: Acids; Algorithms; Analysis; Biology and Life Sciences; Cancer; Cancer genetics; Cell cycle; Classification; Correlation; Cyclin-Dependent Kinase Inhibitor p27 - genetics; Engineering and Technology; Formaldehyde; Gene expression; Gene Expression - genetics; Gene Expression Regulation, Neoplastic - genetics; Genes; Genetic research; Glyceraldehyde-3-phosphate dehydrogenase; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) - genetics; GRB2 Adaptor Protein - genetics; Grb2 protein; Humans; Lung cancer; Lung diseases; Lung Neoplasms - genetics; Medical research; Medicine and Health Sciences; Methods; Neuroendocrine tumors; Neuroendocrine Tumors - genetics; Paraffin; Paraffin Embedding - methods; Pathology; Physical Sciences; Research and Analysis Methods; rhoA GTP-Binding Protein - genetics; RhoA protein; Ribonucleic acid; RNA; RNA, Messenger - genetics; Statistical analysis; Statistical methods; Syntenins - genetics; Technology; Technology application; Tissue Fixation - methods; Tumor markers; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0165181

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. 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). 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. 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.