PURPOSE: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive procedure with a high diagnostic yield for lymph node staging in patients with non-small cell lung cancer (NSCLC). If adequate sampling is achieved, samples obtained by EBUS-TBNA have been shown to be useful for molecular analysis. The purpose of this study was to evaluate the feasibility of simultaneous isolation of DNA, RNA and protein using EBUS-TBNA samples.
METHODS: We extracted DNA, RNA, and protein from 59 archived samples obtained by EBUS-TBNA. There were 14 normal lymph nodes and 45 metastatic lymph nodes in patients with NSCLC. All samples were mixed with Allprotect Tissue Reagent® immediately after taking the biopsy and stored in -80 degrees Celsius for at least one year (12-30 months). We used QIAzol Lysis Reagent® for the sequential extraction of total RNA, DNA, and protein. Commercially available purification kits using column method were used for RNA and DNA isolation. The concentration of RNA and DNA was measured using NanoDrop® and the quality of RNA was evaluated using a 2100 Bioanalyzer®. The concentration of protein was measured by Bradford protein assay.
RESULTS: Total RNA could be successfully extracted in all 59 samples. DNA could be extracted in 58 out of 59 (98.3%) samples and protein could be extracted in 57 out of 59 (96.6%) samples. On average, 7.18ug of total RNA, 7.79ug of DNA, and 3.96mg of protein could be isolated. RNA integrity number (RIN) was measured in 32 samples (11 normal lymph nodes, 21 metastatic lymph nodes) and the average RIN number was 6.2 (2.7-7.3). 20 out of 32 total RNA samples (62.5%) showed a RIN of more than 6, thus meeting the threshold for microarray analysis.
CONCLUSIONS: DNA, RNA and protein can simultaneously be extracted from archived samples obtained by EBUS-TBNA.
CLINICAL IMPLICATIONS: This method facilitates direct comparisons of alterations in the genome, transcriptome, and proteome within metastatic lymph nodes through a minimally invasive approach.
DISCLOSURE: Kazuhiro Yasufuku: Grant monies (from industry related sources): Kazuhiro Yasufuku has received unrestricted grants from Olympus Medical Systems for continuing medical education.
The following authors have nothing to disclose: Takahiro Nakajima, Takashi Anayama, Terumoto Koike, Thomas Waddell, Shaf Keshavjee, Hideki Kimura, Ichiro Yoshino
No Product/Research Disclosure Information