Pcr thermal cycler

Total RNA was extracted from cells and tissues using Qiazol reagent (QIAGEN Sciences #79306, Germany). cDNA was synthesized using the PrimeScript 1^st strand cDNA synthesis kit (TaKaRa Bio., Inc., #6110A, Japan) according to the manufacturer instructions. The TLR4, TLR5, and caveolin-1(Cav1) primer sequences were as follows: β-actin sense TGG AAT CCT GTG GCA TCC ATG AAA C, β-actin anti-sense TAA AAC GCA GCT CAG TAA CAG TCC G; TLR4 sense AGC CTA AGC CAC CTC TCT ACC T, TLR4 anti-sense AGC CTA AGC CAC CTC TCT ACC T; Cav1 human sense GAG CTG AGC GAG AAG CAA GT, Cav1 human anti-sense TCC CTT CTG GTT CTG CAA TC; TLR5 human sense TGC CAC TGT TGA GTG CAA GTC, TLR5 human anti-sense ACC TGG AGA AGC CGA AGG TAA G; Cav1 mouse sense AGC CGC GTC TAC TCC ATC TA, Cav1 mouse anti-sense CAT CAG CAC GCA GAA AGA GA; TLR5 mouse sense GGA CAC TGA AGG ATT TGA AGA TG, TLR5 mouse anti-sense GGA CCA TCT GTA TGC TTG GAA TA. The PCR conditions were: pre-denaturation at 94°C for 3 min followed by 20 (β-actin) and 25 (Cav1, TLR4, and TLR5) cycles of denaturation at 94°C for 30 s, annealing at 58°C (Cav1), 52°C (TLR5) or 59°C (TLR4) for 30 s, extension at 72°C for 30 s, and post-extension at 72°C for 10 min using a TaKaRa PCR Thermal Cycler. PCR products were separated on 3% agarose gel and stained with ethidium bromide.

Quantitative RT-PCR (qRT-PCR) was performed on the Takara PCR thermal cycler using the SYBR Green detection system (Takara, Tokyo, Japan). Cycling conditions consisted of a 3-min hot start at 95°C, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 58–60°C for 10 sec, extension at 72°C for 10 sec, and then a final inactivation at 95°C for 10 sec. Dissociation curve analyses were carried out at the end of the cycling to confirm that one specific product was measured in each reaction. Relative quantification was performed using the ΔΔCT method (27 (link)). Expression normalization was conducted by the expression of GAPDH, a housekeeping gene shown to have stable expression in cancer cell lines (28 (link)). The specific primers for each gene are shown in Table II. All experiments were performed in triplicate.

Total RNA was extracted from A549 and H460 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and was used to synthesize cDNA at 42°C for 1 h using oligo dT primers and AccuPower^® RT-premix (Bioneer Corporation). cDNA was amplified using Tenuto PCR premix (Enzynomics Co., Ltd.) and a PCR thermal cycler (Takara Bio, Inc.) with the following thermocycling conditions: 5 min at 95°C (pre-denaturation), 30 cycles of 20 sec at 94°C, 10 sec at 55°C (for MICA/B) or 65°C (for ULBPs), 30 sec at 72°C and 5 min at 72°C (final extension). The following primer pairs were used: ULBP-1 forward, 5′-GCCAGGATGTCTTGTGAGCA-3′ and reverse, 5′-CAGTGGTGAGTAGACAGGCG-3′; ULBP-2 forward, 5′-CCCTGGGGAAGAAACTAAATGTC-3′ and reverse, 5′-ACTGAACTGCCAAGATCCACTGCT-3′; ULBP-3 forward, 5′-GAGGCTCAGACTGGAACTGG-3′ and reverse, 5′-GCCTCTTCTTCCTGTGCATC-3′; MICA forward, 5′-CAGACTGCCTGCAGGAACTA-3′ and reverse, 5′-TTTCTTCTTACAACAACGGACATA-3′; MICB forward, 5′-CGGACAGACTTTCCATAT GTTT-3′ and reverse, 5′-TCCAACAACAATAAATAAGTG ATG-3′; and β-actin forward, 5′-CATCGTGATGGACTCCGGTGAC-3′ and reverse, 5′-TCAGGTAGTCAGTCAGGTCC-3′. The PCR products were analyzed via 1% agarose gel electrophoresis in TAE buffer with ethidium bromide and a UV illuminator. The densities of the bands were measured using ImageJ v1.53 software (National Institutes of Health).