Simultaneous Isolation of Total RNA, DNA, and Protein Using Samples Obtained by EBUS-TBNA.

Background: : Samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) have been shown to be useful for molecular analysis.
Methods: : The purpose of this study was to evaluate the feasibility of simultaneous isolation of DNA, RNA, and protein using EBUS-TBNA samples. We extracted DNA, RNA, and protein from 59 archived samples obtained by EBUS-TBNA. All samples were mixed with DNA, RNA, and protein-protective solution immediately after taking the biopsy and stored in -80°C for at least 1 year (range, 12 to 30 mo). We used QIAzol Lysis Reagent for the sequential isolation of total RNA, DNA, and protein. The concentration of RNA and DNA was measured and the quality of RNA was evaluated. The concentration of protein was measured using the Bradford protein assay.
Results: : Total RNA was successfully isolated in all 59 samples. DNA was isolated in 58 of 59 (98.3%) samples and protein was isolated in 57 of 59 (96.6%) samples. On average, 7.18 μg of total RNA, 7.79 μg of DNA, and 3.96 mg of protein were isolated. RNA integrity number (RIN) was measured in 32 samples and the average RIN number was 6.2 (range, 2.7 to 7.3). Twenty of 32 total RNA samples (62.5%) showed a RIN of >6.
Conclusions: : DNA, RNA, and protein can simultaneously be isolated from archived samples obtained by EBUS-TBNA. This method facilitates direct comparisons of alterations in the genome, transcriptome, and proteome within metastatic lymph nodes through a minimally invasive approach.