Gtpure ffpe tissue dna extraction kit

Genomic DNA was isolated and purified from formalin-fixed paraffin-embedded tissues using the GTpure FFPE Tissue DNA Extraction Kit (GeneTech, Shanghai, China) in accordance with the manufacturer’s instructions. A fragment method was used to detect Ex19/exon 20 insertion. Mt genes were amplified by polymerase chain reaction. To detect exon 18 mutations (G719X), and exon 21 mutations (L858R and L861Q), the Cycleave method was used based on the basic principle of realtime polymerase chain reaction. Each PCR assay contained forward and reverse primers (each 4 pmol), 2 μl template DNA solution, and 2 units of Hot-Start Taq DNA polymerase (Takara, Shiga Japan) in a 40 ml volume. The PCR conditions consisted of initial denaturation at 95 °C for 3 min; 50 cycles of 95 °C for 15 s, annealing at 56 °C for 30 s and 72 °C for 30 s; and final extension at 72 °C for 5 min. The PCR products were sequenced using the Pyrosequencing PyroMark ID system (Qiagen, Hilden, Germany) following the manufacturer’s instructions.

Genomic DNA (gDNA) from the FFPE sections was extracted using a QuickExtract^™ FFPE DNA Extraction Kit (Cat. QEF81805; Epicentre Biotechnologies, Madison, WI, USA), a QIAamp^® DNA FFPE Tissue Kit (Cat. 56404; Qiagen, Valencia), a Paraffin Sample DNA Extraction Wax-Free^™ DNA Kit (Cat. WF-100; TrimGen, Sparks, MD, USA), and a GTpure^™ FFPE Tissue DNA Extraction Kit (Cat. 536–050; Gene Tech, Shanghai, China), respectively. The manufacturer’s instructions were followed during use of each of the kits. To generate comparative results, each aforementioned kit was used to extract FFPE DNA from two sections mounted on two glass slides or stored in a single tube for each sample. The gDNA of Sus scrofa (pig, porcine), human whole blood, and AsPC-1 cell lines was extracted with the QIAamp^® DNA Blood Mini Kit (Qiagen). DNA concentrations were accurately quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Eugene, OR, USA).