Research Papers:

High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer

Petra Leidinger, Thomas Brefort, Christina Backes, Medea Krapp, Valentina Galata, Markus Beier, Jochen Kohlhaas, Hanno Huwer, Eckart Meese and Andreas Keller _

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Oncotarget. 2016; 7:4611-4623. https://doi.org/10.18632/oncotarget.6566

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Petra Leidinger1,*, Thomas Brefort2,*, Christina Backes3, Medea Krapp2, Valentina Galata3, Markus Beier2, Jochen Kohlhaas2, Hanno Huwer3, Eckart Meese1, Andreas Keller3

1Department of Human Genetics, Saarland University, Homburg, Germany

2The Comprehensive Biomarker Center GmbH, Heidelberg, Germany

3Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany

*These authors contributed equally to this work

Correspondence to:

Andreas Keller, e-mail: [email protected]

Keywords: miRNA, qRT-PCR, NSCLC, diagnosis

Received: August 04, 2015     Accepted: November 09, 2015     Published: December 11, 2015


Validation of biomarkers is essential to advance the translational process to clinical application. Although there exists an increasing number of reports on small non-coding RNAs (microRNAs) as minimally-invasive markers from blood, serum or plasma, just a limited number is verified in follow-up studies. We used qRT-PCR to evaluate a known miRNA signature measured from blood that allowed for separation between patients with non-small cell lung cancer (NSCLC), COPD and healthy controls.

From the data of our previous microarray studies we selected a panel of 235 miRNAs related to lung cancer and COPD. We observed a high concordance between the AUC values of our initial microarray screening and the qRT-PCR data (correlation of 0.704, p < 10–16). Overall, 90.3% of markers were successfully validated. Among the top markers that were concordant between both studies we found hsa-miR-20b-5p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-106a-5p. The qRT-PCR analysis also confirmed that non-small cell lung cancer patients could be accurately differentiated from unaffected controls: a subset of five markers was sufficient to separate NSCLC patients from unaffected controls with accuracy of 94.5% (specificity and sensitivity of 98% and 91%). Beyond differentiation from controls, we also succeeded in separating NSCLC patients from patients with COPD. MiRNAs that were identified as relevant for the separation between lung cancer and COPD by both qRT-PCR and the array-based studies included hsa-miR-26a-5p, hsa-miR-328-3p and hsa-miR-1224-3p. Although for differentiation between NSCLC patients from COPD patients more markers were required, still high accuracy rates were obtained (5 markers: 78.8%; 10 markers: 83.9%; 50 markers: 87.6%).

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