Abi 7500 pcr system

RNAiso Plus reagent (Takara Biotechnology, Dalian, China) was applied for the isolation of total RNA, and reverse-transcription of the isolated RNA to cDNA was performed using an RT Master Mix for qPCR (MedChemExpress, Monmouth Junction, NJ, USA), strictly following the manufacturer’s instructions. Genomic DNA was extracted using a PureLink^TM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA). Real-time quantitative PCR was performed, and results were analyzed using an ABI 7500 PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using SYBR Green qPCR Master Mix (MedChemExpress). The primers used were as follows: HIF-2α, 5′-ATCCCTATGGACGGCGAG-3′ (forward), and 5′-CAACTGCTGCGGGTACTTAT-3′ (reverse); c-Myc, 5′-AAACGACAAGAGGCGGACAC-3′ (forward) and 5′-TGGTCACGCAGGGCAAAA-3′ (reverse); GOT1, 5′-CGAGTACCTGCCCATCCTG-3′ (forward) and 5′-ACCATCGCCCTAAGAAGTCA-3′ (reverse); and β-actin, 5′-CACCCCATTTGATGTTAGTG-3′ (forward) and 5′-CCATTTGCAGTGGCAAAG-3′ (reverse). In addition, PCR was performed on a StepOnePlus^TM Real-Time PCR system to distinguish the wide-type Kras and activated Kras^G12D mutant alleles. Genotyping was confirmed by agarose gel electrophoresis. The primers used were as follows: Krasboth, 5′-AGGCCTGCTGAAAATGACTG-3′ (forward), and 5′-TGGT TCCCTAACACCCAGTT-3′(reverse).

Extraction of total RNA was performed with TRIzol reagent (Tiangen). The RNA was reverse transcribed using the PrimeScript RT reagent kit with gDNA Eraser (TAKARA, RR047A), and the resulting cDNA was amplified by PCR in an ABI-7500 PCR system (Thermo Fisher Scientific) using TB Green Premix Ex Taq II (TAKARA, RR820A). Transcript levels of all target genes were normalized to those of β-actin and are expressed as fold changes relative to the indicated controls, according to the comparative 2^-ΔΔCt value method, where ΔΔ=ΔCt_target-ΔCt_β-actin. The primer sequences (TAKARA) used are listed in Table 1.

A cDNA microarray, which contained seven pancreatic cancer cell lines, and a normal pancreatic duct epithelial cell line (hTERT-HPNE) were obtained from Shanghai Outdo Biotech Company (Shanghai, China). hTERT-HPNE and SW1990 cell lines were maintained in DMEM (Invitrogen, Carlsbad, CA, United States) supplemented with 1% penicillin-streptomycin (Invitrogen) and 10% fetal bovine serum (Invitrogen) at 37°C and 5% CO₂. RNA was extracted from hTERT-HPNE using a TRIzol kit (Sigma-Aldrich Co., St. Louis, MO, United States) according to the manufacturer’s instructions. The extracted RNA was reverse transcribed into cDNA using a PrimeScript RT kit (Takara Bio, Inc., Dalian, China). We then performed qPCR analyses using an ABI 7500 PCR system (Thermo Fisher Scientific, Waltham, MA, United States) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China). β-actin was used as the internal control. The primer sequences used were as follows: MMP11‐forward, 5′‐CTT​GCT​GTA​TCC​CTG​TTG​TG‐3′; MMP11‐reverse, 5′‐ACC​CCT​CCC​CAT​TTG​ACT​G; β-actin‐ forward, 5′‐GAA​GAG​CTA​CGA​GCT​GCC​TGA‐3′; and β-actin‐reverse, 5′‐CAG​ACA​GCA​CTG​TGT​TGG​CG‐3′.