Abstract
RNA-based next-generation sequencing (RNA-seq) represents the gold standard for detecting gene fusion in non-small cell lung cancer (NSCLC). Despite this, RNA instability makes the management of tissue samples extremely complex, resulting in a significant number of test failures with missing data or the need to switch to other techniques. In the present study, we analyzed pre-analytical variables in 140 tumor tissue samples from patients affected by NSCLC to detect features that increase the chances of successful RNA-seq. We found that the success rate of the analysis positively correlates with the RNA concentration and fragmentation index. Interestingly, small biopsies were more suitable samples than surgical specimens and cell blocks. Among surgical specimens, wedge resections demonstrated better results than lobectomy. Moreover, samples stored for less than 30 days (1 month) had a better chance of success than older samples. Defining the role of pre-analytical variables in RNA-seq allows the detection of more suitable samples for analysis and more effective planning of molecular-based diagnostic approaches in NSCLC.