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.

Total RNA was isolated using TRIzol reagent (Invitrogen). The concentration and purity of the RNA were detected with the use of nanodrop (ND-1000, Nanodrop Technologies). cDNA was synthesized with a RevertAid cDNA Synthesis Kit (Thermo Fisher Scientific). The primers designed with Primer Premier 5.0 software were synthesized by Beijing Tsingke Biological Technology (Beijing, China): CDKN2B: F 5′-CCCTGCCACCCTTACCAGA-3′, R 5′-CAGATACCTCGCAATGTCACG-3′; GAPDH: F 5′-TGGATTTGGACGCATTGGTC-3′, R 5′-TTTGCACTGGTACGTGTTGAT-3′. qPCR reactions were performed using Brilliant II SYBR Green qPCR master mix with low ROX (Agilent Technologies, Cedar Creek, TX) and monitored on a Stratagene Mx3000P cycler (Agilent Technologies).

Real‐time multiplex qPCR was performed using a dual‐labelled probe technique with the Quantifast Multiplex PCR + R kit (Qiagen) on a Stratagene Mx3000P Cycler (Agilent Technologies). All reactions were run with the following program: activation at 95°C for 5 min, followed by 40 cycles of two‐step cycling at 95°C for 45 s and 60°C for 45 s. Primers and probes for reference genes (actb, gapdh) and target genes (tlr4) were acquired from Sigma‐Aldrich and are detailed in Table 2. Taqman primers for cxcl8 (Hs00174103_m1) were acquired from Applied Biosystems. Analysis of C_t values was performed using the ΔΔC_t method.

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.