Brilliant ultra fast sybr green qpcr

Patient tissue DNA was obtained by FFPE deparaffinization and a Maxwell 16 CSC DNA FFPE kit (Promega, Madison, WI, USA). The purity, amount, and median size of extracted DNA were measured using a Nanodrop 8000 UV-Vis spectrometer (Thermo Scientific Inc., Wilmington, DE, USA), Qubit 2.0 fluorometer (Life Technologies Inc., Grand Island, NY, USA), and a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA). In addition, the ΔCt value was determined using real-time PCR (Agilent Technologies) with a Mx3005p instrument (Agilent Technologies, USA), FFPE QC kit (Illumina, cat no/ WG-321–1001), and Brilliant Ultra-Fast SYBR Green qPCR (Agilent Technologies, cat no. 600882). DNA was only sequenced if it met our quality criteria: (i) purity: absorption ratio (260 nm/280 nm) >1.8, 260 nm/230 nm >1.8; (ii) total amount >250 ng; (iii) degradation: ΔCt value <2.0, or DNA median size >0.35 kb.

Genomic DNA was extracted from fresh tissue specimens using the QIAamp DNA mini kit (Qiagen, Valencia, CA, USA) or from FFPE tissues using either the Promega Maxwell 16 CSC DNA FFPE kit or the QIAamp DNA FFPE Tissue kit according to the manufacturer’s manual. The purity, amount, and median size of the extracted DNA were measured by the Nanodrop 8000 UV-Vis spectrometer (Thermo Scientific Inc., Wilmington, DE, USA), Qubit 2.0 fluorometer (Life Technologies Inc., Grand Island, NY, USA), and 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA). In addition, ΔCt values were determined using real-time PCR (Agilent Technologies) with Mx3005p instrument (Agilent Technologies, USA), FFPE QC kit (Illumina, cat no. WG-321-1001), and Brilliant Ultra-Fast SYBR Green qPCR (Agilent Technologies, cat no. 600882). If DNA meets the quality criteria such as (i) purity to absorption ratio (260 nm/280 nm) > 1.8, 260 nm/230 nm > 1.8; (ii) total amount > 250 ng; (iii) degradation to ΔCt value < 2.0; or DNA median size > 0.35 kb, it is proceeded onto the sequencing step.