title: High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer.
abstract: 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%).
authors.affiliation: Department of Human Genetics, Saarland University, Homburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Leidinger
authors.affiliation: The Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brefort
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Backes
authors.affiliation: The Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Medea
authors.initials: M
authors.lastname: Krapp
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany.
authors.forename: Valentina
authors.initials: V
authors.lastname: Galata
authors.affiliation: The Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Beier
authors.affiliation: The Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kohlhaas
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany.
authors.forename: Hanno
authors.initials: H
authors.lastname: Huwer
authors.affiliation: Department of Human Genetics, Saarland University, Homburg, Germany.
authors.forename: Eckart
authors.initials: E
authors.lastname: Meese
authors.affiliation: Chair for Clinical Bioinformatics, Saarland University, Saarbrücken, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000235:genetics*; D016022:Case-Control Studies; D015331:Cohort Studies; D005260:Female; D020869:Gene Expression Profiling; D057166:High-Throughput Screening Assays; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics*; D008297:Male; D035683:MicroRNAs / Q000097:blood / Q000235:genetics*; D008875:Middle Aged; D011379:Prognosis; D029424:Pulmonary Disease, Chronic Obstructive / Q000097:blood / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; D035683:MicroRNAs; D012333:RNA, Messenger
keywords: NSCLC; diagnosis; miRNA; qRT-PCR