Amoydx ros1 fusion gene detection kit

ROS1 rearrangements were identified by an AmoyDx^®ROS1 fusion gene detection kit (Amoy Diagnostics Co., Ltd, Xiamen, China). The patterns of ROS1 rearrangements were detected in our study as previously described [9 (link)]. The RT-PCR conditions of cDNA was as follows: one cycle of 95°C for 5 min; followed by 15 cycles of denaturation at 95°C for 25 s, annealing at 64°C for 20 s and elongation at 72°C for 20 s to ensure the specificity; and up to 31 cycles of 93°C for 25 s, 60°C for 35 s (data collection) and 72°C for 20 s. β-actin was used as an internal reference gene to ensure the quality of the extracted RNA and ROS1-rearranged DNA was used as positive control.

The sliced FFPE tumor sections were analyzed by SRL Inc using an in vitro diagnostic AmoyDx ROS1 Fusion Gene Detection Kit (Amoy Diagnostics Co., Ltd) according to manufacturer's protocol. In brief, total RNA was extracted from FFPE tissue using RNeasy FFPE kit according to manufacturer protocol (Qiagen). Reaction samples were mixed with 18.5 μL of ROS1 Reverse Transcription Mixture, 0.5 μL of ROS1 Reverse Transcription Enzyme and 6 μL of total RNA. PCR condition is as follows: 42°C, 1 hour and 95°C, 5 minutes. Complementary DNA (cDNA) were subjected to multiplex RT–PCR as follows: 95°C, 5 minutes, 15 cycles of 95°C, 25 seconds, 64°C, 20 seconds and 72°C, 20 seconds followed by 31 cycles of 93°C, 25 seconds, 60°C, 35 seconds, and 72°C, 20 seconds.

ROS1 was amplified by multiplex real-time PCRs using a Stratagene Mx3000P real-time PCR system (Stratagene, CA) with an AmoyDx^®ROS1 fusion gene detection kit (Amoy Diagnostics Co., Ltd, Xiamen, China) (S2 file). The status of the EGFR mutation and ALK rearrangement were also analyzed by real-time PCR and IHC, according to methods previously described [15 ].