7300 real time pcr system machine

Normal mouse liver was harvested from 25 g C57BL/6 mice and RNA extracted to serve as a comparative normal control. RNA was extracted from RIL-175 cells using miRNeasy mini kit (Qiagen, Germantown, MD, USA). Synthesis of cDNA was performed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD, USA). Real-time PCR was performed using a Perfecta SYBR Green FastMix ROX kit (Quanta Biosciences) with an Applied Biosystems 7300 Real-Time PCR System machine to assess the expression of gastrin and PD-L1. Samples were subjected in triplicate to qRT-PCR, with an initial denaturation step of 95 °C at 3 min, followed by 40 cycles of 95 °C at 15 s and 60 °C at 1 min. The PCR primers used are shown in Table 3. Using in silico primer analysis, some cross-reactivity was noted between the Cckbr primer and murine Zdhhc5 gene that codes for zinc finger DHHC-type palmitoyltransferase 5 that could potentially lead to off-target amplification. However, PCR dissociation curves revealed only one peak consistent with one amplicon (Supplemental Figure S1).

Total RNA (Qiagen, Germantown, MD, USA) was extracted from each of the tumors at necropsy and subjected to qRT-PCR for determination of expression of genes targeted by the siRNAs. The primers designed to analyze the gene expression are shown in Supplementary Table S2. Synthesis of cDNA was performed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD, USA). Real-time PCR was performed using a Perfecta SYBR Green FastMix ROX kit (Quanta Biosciences, Gaithersburg, MD, USA) with an Applied Biosystems 7300 Real Time PCR System machine to assess the expression of GAST and muKRAS. Samples were subjected in triplicate to qRT-PCR or 40 cycles at 60^o C. GAPDH was used as the normalizer for human tumors.