PURPOSE: Diagnosis of lung cancer is achieved by fine needle aspiration (FNA) techniques such as CT-guided FNA and transbronchial needle aspiration (TBNA). The purpose of this study was to establish an optimal storing method for molecular testing in FNA samples.
METHODS: FNA was performed using a 21-gauge needle in surgically resected lung cancer samples. The aspirates were stored according to the following protocol. Group1: The aspirate was flushed out into a collection tube using air and then snap frozen with liquid nitrogen. Group 2: The aspirate was mixed with RNA later® and stored overnight at 4 degrees Celsius. After sample collection for both groups, these samples were stored at -80 Degree Celsius for six months. RNA was extracted from each sample using commercially available RNA extraction kit and the quality and quantity of RNA was measured. Quantitative real-time RT-PCR were performed for human actin beta (hACTB) and keratin 19 (KRT19), and then the result evaluated.
RESULTS: FNA was performed from seven lung cancers. RNA could be extracted from all samples. The median amount of extracted total RNA was 33.9μg for group 1, 35.8μg for group 2. The mean ratio of absorptions at 260nm versus 280nm for the purity of RNA was 2.02 for group 1, 1.97 for group 2. As for the quality of RNA, the mean RNA integrity number (RIN) using a 2100 Bioanalyzer® was 3.5 for group 1, 6.3 for group 2 and the RIN was significantly higher in Group 2 (p=0.0073). RT-PCR for hACTB and KRT19 could be successfully performed in all samples; however, the relative gene expression value showed intra-sample variation depending on the storage method.
CONCLUSIONS: RNA can be extracted from FNA samples and extracted RNA can be used for RT-PCR. However, high quality RNA can only be maintained with RNA later® and the RNA quality may affect mRNA expression analysis using quantitative real-time PCR.
CLINICAL IMPLICATIONS: Samples obtained from CT-guided FNA or TBNA may be utilized for genetic profiling of lung cancer.
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, Thomas Waddell, Shaf Keshavjee, Ichiro Yoshino
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