BACKGROUND: Lung cancer is highly aggressive and tends to metastasize early. Therefore, accurate mediastinal staging is important for therapeutic decision making. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUSTBNA) has emerged as a minimally invasive procedure for mediastinal lymph node sampling and cancer staging. Classical EBUS-TBNA cytology has been combined with molecular staging techniques to improve sensitivity and specificity. This study aimed to assess mRNA integrity in samples acquired by EBUS-TBNA in the clinics. As proof-of-principle experiments, we also investigated whether stable miRNA could be detected in these samples. METHODS: Integrity of mRNA isolated from tumor-positive EBUS-TBNA samples was assessed by calculating the RNA integrity number (RIN). In addition, 4 microRNAs were investigated (miRNA 21, miRNA 155, miRNA 200c, and miRNA 34a) because their relation to lung cancer has been documented recently. A group of patients with benign mediastinal lymphadenopathy served as a control. RESULTS: mRNA isolated from EBUS-TBNA samples was nearly completely degraded if handled under clinical conditions (RIN <5). Intact miRNA was detected in all samples, with no nonspecific amplification in negative control samples. miRNA 21 and miRNA 200c levels were significantly higher in tumor-positive than in control samples (miRNA 21: median, 325,678 [range, 34,822-583,502] vs. 801,430 (range, 17,013-5,362,145]; P<.05; miRNA 200c: median, 9,198 [range, 610-211,121] vs. 42,870 [range, 0-926,252]; P<.05). CONCLUSIONS: Under clinical conditions, mRNA detection is likely unsuitable for improving sensitivity of EBUS-TBNA-facilitated cancer staging. In contrast, detection of miRNA combined with EBUS-TBNA cytology may improve staging sensitivity