Rt7500 real time pcr device

From all study participants, 5 ml of whole blood was obtained (stored in − 80 °C). DNA Blood Mini Kit (Qiagen, Canada) was used to DNA isolation. DNA quality and quantity were evaluated with the use of NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, USA). Genotyping was performed using a real-time PCR method with allele-discriminating software. The Genotyping Master Mix and TaqMan probes (Applied Biosystems, USA) specific for studied TNF-α SNP (Thermo Fisher Scientific, USA, SNP ID: C_7514871_10, cat. no. 4351379) were used to DNA amplification under the condition of manufacturer protocol provided with kit in RT7500 Real-time PCR device (Applied Biosystems, USA). Because we were interested in the assessment of the generalised inflammation process (TNF-α in circulation) instead of local changes (TNF-α in saliva), we decided to use plasma instead of saliva as a test material. The plasma TNF-α level was assessed using TNF alpha Human ELISA Kit Ultrasensitive (Thermo Fisher Scientific, USA, cat. no. KHC3014). The detection range was 0.2–32 pg/mL, and the sensitivity was equal to the minimal detectable dose of this kit (< 0.09 pg/mL).

Prior to starting chemotherapy, 5 mL of whole blood were drawn from all participants and stored in −80 °C until further analyses were performed. DNA Blood Mini Kit (Qiagen, Canada) was used to isolate DNA. The quality and quantity of DNA were assessed using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The evaluation of SNPs of the BIRC5 gene was performed using a real-time PCR method with allelic discrimination software. The Genotyping Master Mix and TaqMan probes (Applied Biosystems, Waltham, MA, USA) specific for studied SNPs (Thermo Fisher Scientific) were used for DNA amplification according to the manufacturer’s protocol in the RT7500 Real-time PCR device (Applied Biosystems, USA). All sample tests were run in triplicate.