Stratagene mx3000p cycler

EGFR mutation detection was conducted using the Amplification‐Refractory Mutation System. FFPE DNA extraction kit (Amoy Diagnostics, Xiamen, China) was used to extract tumor DNA. EGFR mutation was detected by EGFR 29 Mutations Detection Kit (Amoy Diagnostics, Xiamen, China) using 80 ng DNA. The procedures were followed‐up as described in the protocol. PCR was performed on a Stratagene Mx3000P cycler (Agilent, Santa Clara, CA, USA) using the following program: Five minutes at 95°C (one cycle); 25 seconds at 95°C, 20 seconds at 64°C, and 20 seconds at 72°C (15 cycles); 25 seconds at 93°C, 35 seconds at 60°C, and 20 seconds at 72°C (31 cycles). The results were determined as described in the protocol.

Formalin‐fixed paraffin‐embedded (FFPE), fine/core needle aspiration, biopsy, or pleural effusion samples were used for the detection of alterations in the EGFR, KRAS, BRAF, ALK, and ROS1 genes. Genomic DNA and total RNA were extracted from FFPE samples using the AmoyDx FFPE DNA/RNA extraction kit (ADx‐FF03, Amoy Diagnostics, Xiamen, China) according to the manufacturer's recommendations, and from all other samples using the AmoyDx Tissue DNA/RNA extraction kit (ADx‐TI03, Amoy Diagnostics). EGFR, KRAS, and BRAF mutations were detected using 80 ng, 120 ng, and 10 ng of DNA, respectively. ALK and ROS1 rearrangements were detected using 100‐1000 ng of total RNA. The EGFR 29 Mutations Detection Kit (ADx‐EG01), KRAS Mutation Detection Kit (ADx‐KR01), BRAF V600 Mutations Detection Kit (ADx‐BR01), EML4‐ALK Fusion Gene Detection Kit (ADx‐AE01), and ROS1 Gene Fusions Detection Kit (ADx‐RO02) from Amoy Diagnostics were used to detect alterations. For ALK and ROS1 reverse transcription, RNA and 0.5 μL of EA Reverse Transcriptase were added to an EA RT and ROS1 RT Reaction Mix tube, respectively. The samples were then incubated for 1 hour at 42℃ followed by 5 minutes at 95℃. All real‐time qPCRs were performed on a Stratagene Mx3000P^™ cycler (Agilent, Santa Clara, CA, USA) using the following program: 5 minutes at 95℃ (1 cycle); 25 seconds at 95℃, 20 seconds at 64℃, and 20 seconds at 72℃ (15 cycles); 25 seconds at 93℃, 35 seconds at 60℃, and 20 seconds at 72℃ (31 cycles). Ultrapure water was used as negative control, different commercial products were used as positive control for different gene detection, and the conservative sequences of the corresponding gene were used as quality control. Alterations in EGFR, ALK/ROS1, KRAS, and BRAF were defined as Ct values <26, <30, <26, and <28, respectively. For samples positive for ALK and ROS1 rearrangements, DNA sequencing was performed to distinguish the variants as previously described.29, 30

MiRwalk, miRanda and TargetScan were used to predict miR-1246 target genes. The miRNA target genes function were analyzed by the DAVID Bioinformatic Resource (http://david.abcc.ncifcrf.gov/). Based on the potential pathways involved in cell proliferation and apoptosis, four target genes (JARID2, FRK, DR5 and TGFBR3) were selected for RT-PCR analysis after transfection of miR-1246 mimic in H446 cells. The respective primers sequence were obtained from the Primer Bank (http://pga.mgh.harvard.edu/primerbank/). Total RNA was isolated using the E.Z.N.A.^® Total RNA Kit (Omega Bio-Tek, Inc., GA, USA). The expression levels of indicated genes were quantified using RT-PCR analysis. In brief, RNA was transcribed using a QuantiTect reverse-transcription Kit (QIAGEN, Shanghai, China), and the quantitative assessments of cDNA amplification for each gene were detected with a QuantiTect SYBR Green PCR Kit (QIAGEN) by a Stratagene MX3000P Cycler (Agilent). The dissociation curve (melting curve) for each gene was performed to verify the quality of the primes. Gene expression was normalized to the Ct value of b-actin from the same sample and the relative expression level of each mRNA was analyzed using a comparative CT (2^−ΔΔCT) method.